Highly galactosylated anti-tnf-alpha antibodies and uses thereof

ABSTRACT

In one aspect, the disclosure relates to highly galactosylated anti-TNF-alpha antibodies and compositions thereof. In one aspect, the disclosure relates to populations of anti-TNF-alpha antibodies with a high level of galactosylation, and compositions thereof. In one aspect, the disclosure relates to methods of production and use of highly galactosylated anti-TNF-alpha antibodies and populations of anti-TNF-alpha antibodies with a high level of galactosylation. In some embodiments, the anti-TNF-alpha antibody is adalimumab.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/767,117, filed Aug. 11, 2015 which is a national stage filing under35 U.S.C. § 371 of International Patent Application No.PCT/IB2014/000692, filed Feb. 13, 2014, which claims the benefit under35 U.S.C. § 119(e) of U.S. Provisional Application No. 61/764,475,entitled “Highly Galactosylated Anti-TNF-α Antibodies and Uses Thereof,”filed on Feb. 13, 2013, the contents of each of which are incorporatedby reference herein in their entirety.

FIELD OF THE INVENTION

The present invention relates in part to anti-TNF-alpha antibodies.

BACKGROUND OF THE INVENTION

TNF-alpha is a pro-inflammatory cytokine, which plays a protective roleagainst infection and injury in normal immune responses. However,chronically elevated levels of TNF-alpha have been associated withpathogenesis of many autoimmune and inflammatory diseases. A number ofTNF-alpha binding therapeutics have been approved for treatment ofautoimmune and inflammatory diseases including rheumatoid arthritis,psoriasis, Crohn's disease, ankylating spondylitis and ulcerativecolitis. Available therapeutic TNF-alpha binders include antibodies suchas infliximab/Remicade (Centocor) a mouse-human chimeric monoclonal antiTNF-antibody, adalimumab/Humira (Abbott) a fully human anti-TNFantibody, and golimumab/Simponi (Centocor) a fully human anti-TNFantibody. Therapeutic TNF-alpha binders that are antibody-based includeEtanercept/Enbrel (Amgen) a fusion protein of the extracellular domainof TNF-receptor fused to the Fc region of Ig1, and certolizumabpegol/Cimzia (UCB) a Pegylated Fab′ fragment of humanized monoclonalanti-TNF antibody. The therapeutic efficacy of the antibodies andantibody-based TNF-alpha binders varies based on the targeted pathology.In addition, many of the anti-TNF-alpha therapeutics show undesired sideeffects. Anti-TNF alpha antibodies with improved therapeutic propertiesare desired therefore.

SUMMARY OF INVENTION

In one aspect, the disclosure relates to highly galactosylatedanti-TNF-alpha antibodies and compositions thereof. In one aspect, thedisclosure relates to populations of anti-TNF-alpha antibodies with ahigh level of galactosylation, and compositions thereof. In one aspect,the disclosure relates to methods of production and use of highlygalactosylated anti-TNF-alpha antibodies and populations ofanti-TNF-alpha antibodies with a high level of galactosylation.

In one aspect the disclosure provides an anti-TNF-alpha antibody,wherein the antibody is highly galactosylated. In some embodiments, theantibody is highly fucosylated. In some embodiments, the antibodycomprises mono-galactosylated N-glycans. In some embodiments, theantibody comprises bi-galactosylated N-glycans. In some embodiments, theheavy chain of the antibody comprises SEQ ID NO:1, and the light chainof the antibody comprises SEQ ID NO:2. In some embodiments, the antibodyis adalimumab. In some embodiments, the antibody is produced in mammaryepithelial cells of a non-human mammal. In some embodiments, theantibody is produced in a transgenic non-human mammal. In someembodiments, the non-human mammal is a goat, sheep, bison, camel, cow,pig, rabbit, buffalo, horse, rat, mouse or llama. In some embodiments,the non-human mammal is a goat.

In one aspect the disclosure provides compositions of any of theantibodies disclosed herein, wherein the composition further comprisesmilk. In some embodiments, the composition further comprises apharmaceutically-acceptable carrier.

In one aspect the disclosure provides a composition, comprising apopulation of antibodies, wherein the antibody is an anti-TNF-alphaantibody, and wherein the level of galactosylation of the antibodies inthe population is at least 60%. In some embodiments, the level ofgalactosylation of the antibodies in the population is at least 70%. Insome embodiments, the level of galactosylation of the antibodies in thepopulation is at least 80%. In some embodiments, the level offucosylation of the antibodies in the population is at least 50%.

In some embodiments, the level of fucosylation of the antibodies in thepopulation is at least 60%. In some embodiments of any of thecompositions provided herein, the population comprises antibodies thatcomprise mono-galactosylated N-glycans. In some embodiments of any ofthe compositions provided herein, the population comprises antibodiesthat comprise bi-galactosylated N-glycans. In some embodiments of any ofthe compositions provided herein, the ratio of the level ofgalactosylation of the antibodies in the population to the level offucosylation of the antibodies in the population is between 1.0 and 1.4.In some embodiments of any of the compositions provided herein, at least35% of the antibodies in the population comprise bi-galactosylatedN-glycans and at least 25% of the antibodies in the population comprisemono-galactosylated N-glycans. In some embodiments of any of thecompositions provided herein, the heavy chain of the antibody comprisesSEQ ID NO:1, and the light chain of the antibody comprises SEQ ID NO:2.In some embodiments of any of the compositions provided herein, theantibody is adalimumab. In some embodiments of any of the compositionsprovided herein, the antibody is produced in mammary epithelial cells ofa non-human mammal. In some embodiments of any of the compositionsprovided herein, the antibody is produced in a transgenic non-humanmammal. In some embodiments, the non-human mammal is a goat, sheep,bison, camel, cow, pig, rabbit, buffalo, horse, rat, mouse or llama. Insome embodiments, the non-human mammal is a goat. In some embodiments,the composition further comprises milk. In some embodiments, thecomposition further comprises a pharmaceutically-acceptable carrier.

In some embodiments of any of the compositions provided herein, thepopulation of antibodies has an increased level of complement dependentcytotoxicity (CDC) activity when compared to a population of antibodiesnot produced in mammary gland epithelial cells.

In some embodiments of any of the compositions provided herein, thepopulation of antibodies has an increased level of antibody-dependentcellular cytotoxicity (ADCC) activity when compared to a population ofantibodies not produced in mammary gland epithelial cells.

In some embodiments of any of the compositions provided herein, thepopulation of antibodies has an increased ability to suppress TNF-alphaactivity in a subject when compared to a population of antibodies notproduced in mammary gland epithelial cells.

In some embodiments of any of the compositions provided herein, thepopulation of antibodies has an increased ability to bind solubleTNF-alpha when compared to a population of antibodies not produced inmammary gland epithelial cells.

In some embodiments of any of the compositions provided herein, thepopulation of antibodies has an increased ability to bind transmembraneTNF-alpha when compared to a population of antibodies not produced inmammary gland epithelial cells.

In some embodiments of any of the compositions provided herein, thepopulation of antibodies not produced in mammary gland epithelial cellsis produced in cell culture.

In some embodiments of any of the compositions provided herein, thelevel of galactosylation of the antibodies not produced in mammary glandepithelial cells is 50% or lower when compared to the level ofgalactosylation of the antibodies produced in mammary gland epithelialcells. In some embodiments of any of the compositions provided herein,the level of galactosylation of the antibodies not produced in mammarygland epithelial cells is 30% or lower when compared to the level ofgalactosylation of the antibodies produced in mammary gland epithelialcells. In some embodiments of any of the compositions provided herein,the level of galactosylation of the antibodies not produced in mammarygland epithelial cells is 10% or lower when compared to the level ofgalactosylation of the antibodies produced in mammary gland epithelialcells.

In one aspect, the disclosure provides a method for producing apopulation of antibodies, comprising: expressing the population ofantibodies in mammary gland epithelial cells of a non-human mammal suchthat a population of antibodies is produced, wherein the antibody is ananti-TNF-alpha antibody, wherein the level of galactosylation of theantibodies in the population is at least 60%. In some embodiments, themammary gland epithelial cells are in culture and are transfected with anucleic acid that comprises a sequence that encodes the antibody. Insome embodiments, the mammary gland epithelial cells are in a non-humanmammal engineered to express a nucleic acid that comprises a sequencethat encodes the antibody in its mammary gland. In some embodiments, thenucleic acid comprises SEQ ID NO:3 and SEQ ID NO:4. In some embodiments,the mammary gland epithelial cells are goat, sheep, bison, camel, cow,pig, rabbit, buffalo, horse, rat, mouse or llama mammary glandepithelial cells. In some embodiments, the mammary gland epithelialcells are goat mammary gland epithelial cells.

In one aspect, the disclosure provides mammary gland epithelial cellsthat produce any of the antibodies, population of antibodies, orcompositions disclosed herein.

In one aspect, the disclosure provides a transgenic non-human mammalcomprising any of the mammary gland epithelial cells disclosed herein.

In one aspect, the disclosure provides a method comprising administeringany of the antibodies, population of antibodies, or compositionsdisclosed herein to a subject in need thereof. In some embodiments, thesubject has an inflammatory disorder or autoimmune disorder. In someembodiments, the inflammatory disorder or autoimmune disorder isrheumatoid arthritis, psoriasis, Crohn's disease, juvenile idiopathicarthritis, ankylozing spondylitis, ulcerative colitis, chronicinflammation, hepatitis, Behcet's disease, Wegener's granulomatosis, orsarcoidosis.

In one aspect, the disclosure provides a monoclonal anti-TNF antibodycomposition comprising monoclonal anti-TNF antibodies having glycanstructures on the Fc glycosylation sites (Asn297, EU numbering), whereinsaid glycan structures have a galactose content of more than 60%.

In one aspect, the disclosure relates to an anti-TNF-alpha antibody,wherein the antibody contains an oligomannose. In some embodiments, theheavy chain of the antibody comprises SEQ ID NO:1, and the light chainof the antibody comprises SEQ ID NO:2. In some embodiments, the antibodyis adalimumab.

In some embodiments, the antibody is produced in mammary epithelialcells of a non-human mammal. In some embodiments, the antibody isproduced in a transgenic non-human mammal. In certain embodiments, thenon-human mammal is a goat, sheep, bison, camel, cow, pig, rabbit,buffalo, horse, rat, mouse or llama. In certain embodiments, thenon-human mammal is a goat.

In some embodiments, an anti-TNF-alpha antibody that contains anoligomannose further comprising milk.

In one aspect, the disclosure relates to a composition comprising ananti-TNF-alpha antibody that contains an oligomannose, furthercomprising a pharmaceutically-acceptable carrier.

In one aspect, the disclosure relates to a composition, comprising: apopulation of antibodies, wherein the antibody is an anti-TNF-alphaantibody, and wherein the at least 30% of the antibodies contain atleast one oligomannose. In some embodiments, the antibodies exhibit ahigh mannose glycosylation pattern. In some embodiments, at least onechain of the milk-derived antibodies contains an oligomannose and isnon-fucosylated. In some embodiments, the major carbohydrate of themilk-derived antibodies is non-fucosylated.

In some embodiments, the heavy chain of the antibody within thecomposition comprises SEQ ID NO:1, and the light chain of the antibodycomprises SEQ ID NO:2. In some embodiments, the antibody is adalimumab.In some embodiments, the antibody is produced in mammary epithelialcells of a non-human mammal. In some embodiments, the antibody isproduced in a transgenic non-human mammal. In some embodiments, thenon-human mammal is a goat, sheep, bison, camel, cow, pig, rabbit,buffalo, horse, rat, mouse or llama. In certain embodiments, thenon-human mammal is a goat.

In some embodiments, the composition further comprises milk. In someembodiments, the composition further comprises apharmaceutically-acceptable carrier.

In some embodiments, the population of antibodies within the compositionhas an increased level of complement dependent cytotoxicity (CDC)activity when compared to a population of antibodies not produced inmammary gland epithelial cells.

In some embodiments, the population of antibodies within the compositionhas an increased level of antibody-dependent cellular cytotoxicity(ADCC) activity when compared to a population of antibodies not producedin mammary gland epithelial cells. In some embodiments, the populationof antibodies not produced in mammary gland epithelial cells is producedin cell culture.

In some embodiments wherein an antibody contains an oligomannose, theoligomannose is Man5. In some embodiments wherein an antibody containsan oligomannose, the oligomannose is Man6. In some embodiments whereinan antibody contains an oligomannose, the oligomannose is Man7. In someembodiments wherein a composition comprises an antibody that contains anoligomannose, the oligomannose is Man5. In some embodiments wherein acomposition comprises an antibody that contains an oligomannose, theoligomannose is Man6. In some embodiments wherein a compositioncomprises an antibody that contains an oligomannose, the oligomannose isMan7.

BRIEF DESCRIPTION OF THE DRAWINGS

The drawings are first described. It is to be understood that thedrawings are exemplary and not required for enablement of the invention.

FIG. 1 shows a representative oligosaccharide signature of N-glycans ofa population of highly galactosylated adalimumab antibodies from goat#1.

FIG. 2 shows an oligosaccharide signature of N-glycans of a populationof highly galactosylated adalimumab antibodies from goat #1 at day 7 oflactation.

FIG. 3 shows an oligosaccharide signature of N-glycans of a populationof highly galactosylated adalimumab antibodies from goat #1 at day 17 oflactation.

FIG. 4 shows an oligosaccharide signature of N-glycans of a populationof highly galactosylated adalimumab antibodies from goat #1 at day 32 oflactation.

FIGS. 5A and 5B show a summary of the percentages of N-glycanoligosaccharides of populations of a highly galactosylated adalimumabantibodies from goat #1 at various days of lactation.

FIG. 6 shows an oligosaccharide signature of N-glycans of a populationof highly galactosylated adalimumab antibodies from goat #2 at day 3 ofhormone induced lactation.

FIG. 7 shows an oligosaccharide signature of N-glycans of a populationof highly galactosylated adalimumab antibodies from goat #2 at day 11 ofhormone induced lactation.

FIG. 8 shows an oligosaccharide signature of N-glycans of a populationof highly galactosylated adalimumab antibodies from goat #2 at day 21 ofhormone induced lactation.

FIGS. 9A and 9B show a summary of the percentages of N-glycanoligosaccharides of populations of a highly galactosylated adalimumabantibodies from goat #2 at various days of hormone induced lactation.

FIG. 10 shows that a transgenically produced adalimumab antibody bindssoluble TNF-alpha.

FIG. 11 shows that a transgenically produced adalimumab antibody bindsto CD16 expressed on NK cells as shown by a competition assay with theanti-CD16 antibody 3G8.

FIG. 12 shows that a transgenically produced adalimumab antibody bindsboth soluble TNF-alpha and Jurkat expressing CD16 cells while anaglycosylated version of the transgenically produced adalimumab antibodydoes not.

FIGS. 13A-13D show a summary of predominant glycan forms present inpopulations of transgenically produced adalimumab antibodies from ninedifferent goats #1-9.

FIGS. 14A and 14B show a summary of the percentages of N-glycanoligosaccharides of populations of transgenically produced adalimumabantibodies from goat #10 and goat #1 during hormone induced lactation.

FIGS. 15A and 15B show a summary of N-glycan oligosaccharides ofpopulations of transgenically produced adalimumab antibodies from eightdifferent goats, goats #2-9.

FIG. 16 shows antigenic recognition of transgenically producedadalimumab (“TG-adalimumab”) and Humira antibodies on membrane TNF-αtransfected Jurkat cells clone 2B3. The results shown are expressed asMFI and derived from an average of 3 experiments. Mean+/−SEM.

FIG. 17 shows binding of anti-TNF-α antibodies on CD16 expressed by NKcells in a competition assay. The mean fluorescence values (MFI)observed are expressed as the percent binding, where 100% is anarbitrary value corresponding to maximum 3G8 binding observed withoutthe tested antibodies and 0% corresponds to the MFI in the absence ofthe antibody 3G8. Mean of 3 experiments+/−SEM.

FIG. 18 shows binding of anti-TNF-α antibodies on FcRn expressed byFcRn-transfected Jurkat cells in a competition assay with Alexa 488coupled-Rituximab antibody. The mean fluorescence values (MFI) observedare expressed as the percent binding (100% is an arbitrary valuecorresponding to maximum Rituximab-A488 binding alone), as a function ofattested antibody concentration. Mean of 3 experiments+/−SEM.

FIG. 19 shows CDC activity of the anti TNF-α antibodies on membraneTNF-α transfected Jurkat cells clone 2B3 (mean of 4 assays). Results areexpressed as percent of lysis of mbTNF-α Jurkat cells as a function ofantibody concentration. Mean+/−SEM.

FIG. 20 shows neutralization of TNF-α-mediated cytotoxicity in L929cells by anti-TNF-α antibodies. Results are expressed as percent ofneutralization as a function of antibody concentration. Mean of 4assays+/−SEM.

FIG. 21 shows percent lysis (in arbitrary units) mediated bytransgenically produced adalimumab compared to Humira.

FIG. 22 presents an inhibition curve showing CD16 binding activity oftransgenically produced adalimumab from nine different goats compared toHumira.

FIG. 23 presents IC50 values associated with CD16 binding activity oftransgenically produced adalimumab from nine different goats and Humiraas assessed by a competitive assay using NK cells.

FIG. 24 shows CDC activity of transgenically produced adalimumab fromnine different goats compared to Humira.

FIG. 25 shows EC50 values (ng/ml) associated with CDC activity oftransgenically produced adalimumab from nine different goats compared toHumira.

DETAILED DESCRIPTION OF INVENTION

In one aspect, the disclosure provides anti-TNF-alpha antibodies whereinthe antibody is highly galactosylated. Anti-TNF-alpha antibodies bindTNF-alpha and have been used as a therapeutic in a variety of diseasescharacterized by dysregulation of TNF-alpha, including inflammatorydisorders. In some embodiments, the anti-TNF-alpha antibody that ishighly galactosylated is infliximab/Remicade (Centocor),adalimumab/Humira (Abbott), or golimumab/Simponi (Centocor). In someembodiments, the anti-TNF-alpha antibody that is highly galactosylatedis adalimumab.

In some embodiments, the anti-TNF-alpha antibody that is highlygalactosylated includes a heavy chain which comprises SEQ ID NO:1. Insome embodiments, the anti-TNF-alpha antibody that is highlygalactosylated includes a light chain which comprises SEQ ID NO:2. Insome embodiments, the anti-TNF-alpha antibody that is highlygalactosylated includes a heavy chain which comprises SEQ ID NO:1 and alight chain which comprises SEQ ID NO:2. In some embodiments, theanti-TNF-alpha antibody that is highly galactosylated includes a heavychain which consists of SEQ ID NO:1. In some embodiments, theanti-TNF-alpha antibody that is highly galactosylated includes a lightchain that consists of SEQ ID NO:2. In some embodiments, theanti-TNF-alpha antibody that is highly galactosylated includes a heavychain which consists of SEQ ID NO:1 and a light chain that consists ofSEQ ID NO:2. In some embodiments, the anti-TNF-alpha antibody that ishighly galactosylated is adalimumab.

The heavy chain of adalimumab is provided in SEQ ID NO:1:

MEFGLSWLFLVAILKGVQCEVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAITWNSGHIDYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGK

The light chain of adalimumab is provided in SEQ ID NO:2:

MDMRVPAQLLGLLLLWLRGARCDIQMTQSPSSLSASVGDRVTITCRASQGIRNYLAWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQRYNRAPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

The sequences are based on the sequences of adalimumab published in U.S.Pat. No. 6,090,382. In some embodiments, the sequences of adalimumab arethose as published in U.S. Pat. No. 6,090,382.

It should further be appreciated that in some embodiments, thedisclosure also includes antibodies that are based on the sequence ofadalimumab but that include mutations that provide the antibodies withadditional beneficial desired properties related to bioavailability,stability etc.

In one aspect, the disclosure provides highly galactosylatedanti-TNF-alpha antibodies that can be used in anti-inflammatorytreatment. In some embodiments, the anti-TNF-alpha antibody that ishighly galactosylated is infliximab/Remicade (Centocor),adalimumab/Humira (Abbott), or golimumab/Simponi (Centocor). In someembodiments, the anti-TNF-alpha antibody is adalimumab. In someembodiments, the anti-TNF-alpha antibodies can be used in the treatmentof inflammatory disorders and the treatment of autoimmune diseases. Asshown herein, the highly galactosylated anti-TNF-alpha antibodies aresurprisingly effective in their anti-inflammatory activity because oftheir enhanced ADCC and CDC activities (as compared to non-highlygalactosylated antibodies). However, it should appreciated that thehighly galactosylated antibodies anti-TNF-alpha disclosed herein mayinclude additional modifications to further increase theiranti-inflammatory activity.

Anti-inflammatory activity of intravenous administered Ig can resultfrom a subset of the IgG molecules that have terminal alpha-2,6-sialicacid linkages on their Fc-linked glycans. The anti-inflammatory activityof a population of the intravenous administered Ig was increased byintroducing a terminal alpha-2,6-sialic acid linkage on all Fc-linkedglycans (See e.g., Anthony et al., Identification of a receptor requiredfor the anti-inflammatory activity of IVIG, PNAS 105: 19571-19578,2008).

In one aspect, the disclosure provides highly galactosylatedanti-TNF-alpha antibodies that also have 2,6 sialylated Fc glycans. Thesialylation of the galactosylated anti-TNF alpha antibodies is believedto synergistically increase the anti-inflammatory action of the highlygalactosylated anti-TNF-alpha antibodies.

In one aspect, the highly galactosylated anti-TNF-alpha antibodiesdisclosed herein are generated by producing the antibody in a transgenicmammal or mammary epithelial cells. As shown herein, highlygalactosylated anti-TNF-alpha antibodies generated by in a transgenicmammal or mammary epithelial cells are not fully sialylated. Thesialylation levels of such antibodies can be increased for instance bysubjecting the antibodies to sialyl transferases. The antibodies can besubjected to sialyl transferases in vitro or in vivo. Highlygalactosylated anti-TNF-alpha antibodies can be sialylated in vitro bysubjecting the purified or partially antibody to a sialyl transferaseand the appropriate saccharide based substrate. Highly galactosylatedanti-TNF-alpha antibodies can be sialylated in vivo by producing asialyl transferase in the mammary gland or mammary epithelial cells.

In one aspect, the disclosure provides methods for the production in themammary gland of transgenic animals and mammary epithelial cells ofhighly galactosylated anti-TNF-alpha antibodies, with increased levelsof alpha-2,6-sialylation. Thus, the methods provided herein allow forthe production in the mammary gland of transgenic animals and mammaryepithelial cells of highly galactosylated anti-TNF-alpha antibodies withincreased anti-inflammatory properties. It should be appreciated thatthe methods provided herein to increase the anti-inflammatory propertiesof highly galactosylated anti-TNF-alpha antibodies can be applied to anyanti-TNF-alpha antibody, thereby providing anti-inflammatory highlygalactosylated anti-TNF-alpha antibodies with synergistic mode ofactions.

In one aspect, the disclosure provides transgenic animals (and mammaryepithelial cells) that are transgenic for the production in the mammarygland of anti-TNF-alpha antibodies and that are transgenic for theproduction of sialyl transferase. The therapeutic antibodies produced bysuch animals and cells are expected to be highly galactosylated and haveincreased levels of terminal alpha-2,6-sialic acid linkages on theirFc-linked glycans. In some embodiments, the transgenic animals (andmammary epithelial cells) are transgenic for the production in themammary gland of an exogenous anti-TNF-alpha antibody and are transgenicfor the production of sialyl transferase. In some embodiments, theanti-TNF-alpha antibody is adalimumab.

In one aspect, the disclosure provides methods of treating inflammationor autoimmune disease in a subject comprising administering to a subjectthe highly galactosylated anti-TNF-alpha antibodies that have increasedlevels of terminal alpha-2,6-sialic acid linkages on their Fc-linkedglycans.

In one aspect, the disclosure provides anti-TNF-alpha antibodies whereinthe antibody is highly galactosylated. In some embodiments, thedisclosure provides anti-TNF-alpha antibodies, wherein the antibody ishighly fucosylated. In some embodiments, the disclosure providesanti-TNF-alpha antibodies, wherein the antibody is highly galactosylatedand highly fucosylated. In some embodiments, the disclosure providesanti-TNF-alpha antibodies, wherein the antibody is highly galactosylatedand highly fucosylated and has terminal sialic acid moieties on the Fcglycans. In some embodiments, the highly galactosylated antibodycomprises one or more mono-galactosylated N-glycans. In someembodiments, the highly galactosylated antibody comprisesbi-galactosylated N-glycans.

In one aspect, the disclosure provides a monoclonal anti-TNF antibodycomposition comprising monoclonal antibodies having on the Fcglycosylation sites (Asn 297, EU numbering) glycan structures, whereinsaid glycan structures have a galactose content more than 60%. In oneembodiment the anti-TNF monoclonal antibodies are purified. The “EUnumbering system” or “EU index” is generally used when referring to aresidue in an immunoglobulin heavy chain constant region (e.g., the EUindex reported in Kabat et al., Sequences of Proteins of ImmunologicalInterest, 5th ed., Public Health Service, National Institutes of Health,Bethesda, Md. (1991) expressly incorporated herein by reference). Thetypical glycosylated residue position in an antibody is the asparagineat position 297 according to the EU numbering system (“Asn297”).

It should be appreciated that any of the anti-TNF monoclonal antibodiesdisclosed herein may be partially or completely purified.

Antibodies can be glycosylated with an N-glycan at the Fc-gammaglycosylation site in the heavy chain (Asn297) of the Fc region.Generally, antibodies include two heavy chains and each antibodytherefore can have two Fc-gamma N-glycans. A variety of glycosylationpatterns have been observed at the Fc gamma glycosylation site and theoligosaccharides found at this site include galactose,N-acetylglucosamine (GlcNac), mannose, sialic acid, N-acetylneuraminicacid (NeuAc or NANA), N-glycolylneuraminic (NGNA) and fucose. N-glycansfound at the Fc gamma glycosylation site generally have a common corestructure consisting of an unbranched chain of a firstN-acetylglucosamine (GlcNAc), which is attached to the asparagine of theantibody, a second GlcNAc that is attached to the first GlcNac and afirst mannose that is attached to the second GlcNac. Two additionalmannoses are attached to the first mannose of the GlcNAc-GlcNAc-mannosechain to complete the core structure and providing two “arms” foradditional glycosylation. In addition, fucose residues may be attachedto the N-linked first GlcNAc.

The two arm core structure is also referred to as the “antenna”. Themost common type of glycosylation of the “arms” of the N-glycan motifsfound in plasma antibodies is of the complex type, i.e., consisting ofmore than one type of monosaccharide. In the biosynthetic route to thisN-glycan motif, several GlcNAc transferases attach GlcNAc residues tothe mannoses of the glycan core, which can be further extended bygalactose, sialic acid and fucose residues. This glycosylation motif iscalled “complex” structure.

A second glycosylation motif found on the “arms” of the N-glycan corestructure is a “high-mannose” motif, which is characterized byadditional mannoses (attached either as branched or unbranded chains).

A third glycosylation motif is a hybrid structures in which one of thearms is mannose substituted while the other arm is complex.

A “galactosylated” antibody, as used herein, refers to any antibody thathas at least one galactose monosaccharide in one of its N-glycans.Galactosylated antibodies include antibodies where the two N-glycanseach have complex type motifs on each of the arms of the N-glycanmotifs, antibodies where the two N-glycans have a complex type motif ononly one of the arms of the N-glycan motifs, antibodies that have oneN-glycan with complex type motifs on each of the arms of the N-glycan,and antibodies that have one N-glycan with a complex type motif on onlyone of the arms of the N-glycan motifs. Antibodies that include at leastone galactose monosaccharide include antibodies with N-glycans such asG1 (one galactose), G1F (one galactose, one fucose), G2 (two galactoses)and G2F (two galactoses, one fucose). In addition, the N-glycan thatincludes at least one galactose monosaccharide can be sialylated or notsialylated. It should further be appreciated that the N-glycans may alsocontain additional galactose residues, such as alpha-Gal, in one or morearms of the complex glycan motif, potentially resulting in an N-glycanwith four galactose moieties.

A “highly galactosylated” antibody, as used herein, refers to anantibody that includes at least two galactose monosaccharides in theN-glycan motifs. Highly galactosylated antibodies include antibodieswhere the two N-glycans each have complex type motifs on each of thearms of the N-glycan motifs, antibodies where the two N-glycans have acomplex type motif on only one of the arms of the N-glycan motifs, andantibodies that have one N-glycan with a complex type motif on each ofthe arms of the N-glycan. Thus, highly galactosylated antibodies includeantibodies in which both N-glycans each include one galactose in theglycan motif (e.g., G1 or G1F), antibodies that include at least oneN-glycan with two galactoses in the glycan motif (e.g., G2 or G2F), andantibodies with 3 or 4 galactoses in the glycan motif (e.g., (i) oneN-glycan with a G1 glycan motif and one N-glycan with a G2 or G2F glycanmotif or (ii) two N-glycan with G2 or G2F). In some embodiments, thehighly galactosylated antibody includes at least three galactosemonosaccharides in the glycan motifs. In some embodiments, the highlygalactosylated antibody includes at least four galactose monosaccharidesin the glycan motifs.

In some embodiments the glycosylation exhibits a high mannoseglycosylation pattern. As used herein, a “high mannose glycosylationpattern” is intended to refer to an antibody that contains at least oneoligomannose or a composition of antibodies wherein at least 30% of theantibodies contain at least one oligomannose. In some embodiments atleast 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of the carbohydrates ofthe antibodies are oligomannose. In some embodiments at least 30%, 40%,50%, 60%, 70%, 80%, 90% or more of the carbohydrates of the antibodiesare non-fucosylated oligomannose. In other embodiments less than 50%,40%, 30%, 20%, 10%, 5% or fewer of the carbohydrates of the antibodiesare fucose-containing. In still other embodiments the antibodies are lowin fucose and high in oligomannose. Therefore, in further embodiments atleast 30%, 40%, 50%, 60%, 70%, 80% or 90% or more of the carbohydratesof the antibodies are oligomannose and less than 50%, 40%, 30%, 20%, 10%or 5% of the carbohydrates of the antibodies are fucose-containing.Therefore, in yet a further embodiment at least 30%, 40%, 50%, 60%, 70%,80% or 90% or more of the carbohydrates of the antibodies arenon-fucosylated oligomannose and less than 50%, 40%, 30%, 20%, 10% or 5%of the carbohydrates of the antibodies are fucose-containing.

In some aspects, the mannose-containing oligosaccharides range from Man5to Man9, with the number indicating the number of mannose residues. Forexample, mannose-containing oligosaccharides can include Man5, Man6,Man7, Man8 and Man9. In certain embodiments, transgenically producedadalimumab exhibits a high Man6 content, as revealed by goats 2, 4 and 5described herein. In some embodiments, the major carbohydrate intransgenically produced adalimumab is Man5. In some embodiments, atleast 10%, 15%, or more of the carbohydrates in transgenically producedadalimumab are Man5. Advantageously, at least 20% of the carbohydratesin transgenically produced adalimumab are Man5. In other embodiments,the major carbohydrate in transgenically produced adalimumab is Man6. Insome embodiments, at least 10%, 15%, or more of the carbohydrates intransgenically produced adalimumab are Man6. Advantageously, at least20% of the carbohydrates in transgenically produced adalimumab are Man6In other embodiments, the major carbohydrate in transgenically producedadalimumab is Man7. In some embodiments, at least 10%, 15%, or more ofthe carbohydrates in transgenically produced adalimumab are Man7.Advantageously, at least 20% of the carbohydrates in transgenicallyproduced adalimumab are Man7.

The glycosylation pattern of the N-glycans can be determined by avariety of methods known in the art. For example, methods of analyzingcarbohydrates on proteins have been described in U.S. PatentApplications US 2006/0057638 and US 2006/0127950. The methods ofanalyzing carbohydrates on proteins are incorporated herein byreference.

In some embodiments, the highly galactosylated antibody is produced inmammary epithelial cells of a non-human mammal. In some embodiments, theantibody is produced in a transgenic non-human mammal. In someembodiments, the non-human mammal is a goat, sheep, bison, camel, cow,pig, rabbit, buffalo, horse, rat, mouse or llama. In some embodiments,

the non-human mammal is a goat.

In some embodiments, the highly glycosylated antibody is produced incells other than in mammary epithelial cells of a non-human mammal. Insome embodiments, the antibody is produced in cells other than inmammary epithelial cells of a non-human mammal and modified afterproduction to increase the number of galactose groups on the N-glycan(e.g., through the action of enzymes such as transferases).

In one aspect, the disclosure provides compositions comprising highlygalactosylated antibodies. In some embodiments, the compositioncomprising highly galactosylated antibodies further comprises milk. Insome embodiments, the composition comprising highly galactosylatedantibodies further comprises a pharmaceutically-acceptable carrier.

In one aspect, the disclosure provides compositions comprisingmonoclonal anti-TNF antibody compositions having on the Fc glycosylationsites (Asn 297, EU numbering) glycan structures, wherein said glycanstructures of the monoclonal antibodies have a galactose content morethan 60%. In some embodiments, the composition comprising monoclonalanti-TNF antibody compositions further comprises milk. In someembodiments, the composition comprising monoclonal anti-TNF antibodycompositions further comprises a pharmaceutically-acceptable carrier.

Populations of Antibodies

In one aspect, the disclosure provides a composition comprising apopulation of antibodies, wherein the antibody is an anti-TNF-alphaantibody, and wherein the level of galactosylation of the antibodies inthe population is at least 60%. In some embodiments, the level ofgalactosylation of the antibodies in the population is at least 70%. Insome embodiments, the level of galactosylation of the antibodies in thepopulation is at least 80%. In some embodiments, the level offucosylation of the antibodies in the population is at least 50%. Insome embodiments, the level of fucosylation of the antibodies in thepopulation is at least 60%. In some embodiments, the populationcomprises antibodies that comprise mono-galactosylated N-glycans. Insome embodiments, the population comprises antibodies that comprisebi-galactosylated N-glycans. In some embodiments, the ratio of the levelof galactosylation of the antibodies in the population to the level offucosylation of the antibodies in the population is between 1.0 and 1.4.In some embodiments, at least 35% of the antibodies in the populationcomprise bi-galactosylated N-glycans and at least 25% of the antibodiesin the population comprise mono-galactosylated N-glycans. In someembodiments, the level of sialylation in the antibodies is at least 50%.In some embodiments, the level of sialylation in the antibodies is atleast 70%. In some embodiments, the level of sialylation in theantibodies is at least 90%. In some embodiments, the antibodies arefully sialylated.

In some embodiments, the anti-TNF-alpha antibody of the populations ofantibodies with a high level of galactosylation is infliximab/Remicade(Centocor), adalimumab/Humira (Abbott), or golimumab/Simponi (Centocor).In some embodiments, the anti-TNF-alpha antibody of the populations ofantibodies with a high level of galactosylation is adalimumab. In someembodiments, the anti-TNF-alpha antibody of the populations ofantibodies with a high level of galactosylation comprises a heavy chainwhich comprises SEQ ID NO:1. In some embodiments, the anti-TNF-alphaantibody of the populations of antibodies with a high level ofgalactosylation comprises a light chain which comprises SEQ ID NO:2. Insome embodiments, the anti-TNF-alpha antibody of the populations ofantibodies with a high level of galactosylation comprises a heavy chainwhich comprises SEQ ID NO:1 and a light chain which comprises SEQ IDNO:2. In some embodiments, the anti-TNF-alpha antibody of thepopulations of antibodies with a high level of galactosylation comprisesa heavy chain which consists of SEQ ID NO:1. In some embodiments, theanti-TNF-alpha antibody of the populations of antibodies with a highlevel of galactosylation comprises a light chain that consists of SEQ IDNO:2. In some embodiments, the anti-TNF-alpha antibody of thepopulations of antibodies with a high level of galactosylation comprisesa heavy chain which consists of SEQ ID NO:1 and a light chain thatconsists of SEQ ID NO:2. In some embodiments, the anti-TNF-alphaantibody of the populations of antibodies with a high level ofgalactosylation is adalimumab.

The biosynthesis of N-glycans is not regulated by a template, as is thecase with proteins, but is mainly dependent on the expression andactivity of specific glycosyltransferases in a cell. Therefore, aglycoprotein, such as an antibody Fc domain, normally exists as aheterogeneous population of glycoforms which carry different glycans onthe same protein backbone.

A population of anti-TNF-alpha antibodies that is highly galactosylatedis a population of antibodies wherein the level of galactosylation ofthe antibodies in the population is at least 50%, at least 60%, at least70%, at least 80%, at least 90%, up to 100% of galactosylation. In someembodiments of the population of antibodies that is highlygalactosylated, the level of galactosylation of the antibodies in thepopulation is at least 60%.

The level of galactosylation as used herein is determined by thefollowing formula:

$\sum\limits_{l = 1}^{n}\left( {\frac{\left( {{number}\mspace{14mu} {of}\mspace{14mu} {Gal}} \right.}{\left( {{number}\mspace{14mu} {of}\mspace{14mu} A} \right)}*\left( {\% \mspace{14mu} {relative}\mspace{14mu} {Area}} \right)} \right)$

wherein:

-   -   n represents the number of analyzed N-glycan peaks of a        chromatogram, such as a Normal-Phase High Performance Liquid        Chromatography (NP HPLC) spectrum    -   “number of Gal” represents the number of Galactose motifs on the        antennae of the glycan corresponding to the peak, and    -   “number of A” corresponds to the number of N-acetylglucosamine        motifs on the antennae of the glycan form corresponding to the        peak (excluding the two N-acetylglucosamine motifs of the core        structure), and    -   “% relative Area” corresponds to % of the Area under the        corresponding peak

The level of galactosylation of antibodies in a population of antibodiescan be determined, for instance, by releasing the N-glycans from theantibodies, resolving the N-glycans on a chromatogram, identifying theoligosaccharide motif of the N-glycan that corresponds to a specificpeak, determining the peak intensity and applying the data to theformula provided above.

Anti-TNF-alpha antibodies that are galactosylated include antibodiesthat are mono-galactosylated N-glycans and bi-galactosylated N-glycans.

In some embodiments of the population of antibodies that are highlygalactosylated, the population comprises antibodies that comprisemono-galactosylated N-glycans, which may or may not be sialylated. Insome embodiments of the population of antibodies that is highlygalactosylated, at least 1%, at least 5%, at least 10%, at least 15%, atleast 20%, at least 25%, at least 30%, at least 40%, at least 50%, atleast 60%, at least 70%, at least 80%, at least 90%, up to 100% of theantibody N-glycans comprise mono-galactosylated N-glycans. In someembodiments of the population of antibodies that is highlygalactosylated, at least 25% of the antibodies comprisemono-galactosylated N-glycans.

In some embodiments of the population of antibodies that are highlygalactosylated, the population comprises antibodies that comprisebi-galactosylated N-glycans, which may or may not be sialylated. In someembodiments of the population of antibodies that is highlygalactosylated, at least 1%, at least 5%, at least 10%, at least 15%, atleast 20%, at least 25%, at least 30%, at least 40%, at least 50%, atleast 60%, at least 70%, at least 80%, at least 90%, up to 100% of theantibody N-glycans comprise bi-galactosylated N-glycans. In someembodiments of the population of antibodies that is highlygalactosylated, at least 35% of the antibodies comprisebi-galactosylated N-glycans.

In some embodiments of the population of antibodies that is highlygalactosylated, the population comprises antibodies that comprisemono-galactosylated N-glycans, which may or may not be sialylated, andantibodies that comprise bi-galactosylated N-glycans, which may or maynot be sialylated. In some embodiments of the population of antibodiesthat is highly galactosylated, at least 1%, at least 5%, at least 10%,at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, atleast 50%, at least 60%, at least 70%, at least 80%, at least 90%, up to99% of the antibody N-glycans comprise mono-galactosylated N-glycans,and at least 1%, at least 5%, at least 10%, at least 15%, at least 20%,at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, atleast 70%, at least 80%, at least 90%, up to 99% of the antibodyN-glycans comprise bi-galactosylated N-glycans. In some embodiments ofthe population of antibody N-glycans that is highly galactosylated, atleast 25% of the antibody N-glycans comprise mono-galactosylatedN-glycans and at least 35% of the antibodies comprise bi-galactosylatedN-glycans.

In some embodiments of the population of antibodies that is highlygalactosylated, the population comprises antibodies that are highlyfucosylated. A population of antibodies that is highly fucosylated is apopulation of antibodies wherein the level of fucosylation of theantibody N-glycans in the population is at least 40%, at least 50%, atleast 60%, at least 70%, at least 80%, at least 90%, up to 100% offucosylation. In some embodiments in the population of antibodies thatis highly galactosylated, the level of fucosylation of the antibodyN-glycans is at least 50%.

The level of fucosylation as used herein is determined by the followingformula:

$\sum\limits_{i = 1}^{n}{\left( {{number}\mspace{14mu} {of}\mspace{14mu} {Fucose}} \right)*\left( {\% \mspace{14mu} {relative}\mspace{14mu} {Area}} \right)}$

wherein:

-   -   n represents the number of analyzed N-glycan peaks of a        chromatogram, such as a Normal-Phase High Performance Liquid        Chromatography (NP HPLC) spectrum, and    -   “number of Fucose” represents the number of Fucose motifs on the        glycan corresponding to the peak, and    -   “% relative Area” corresponds to % of the Area under the        corresponding peak containing the Fucose motif.

Antibodies that are fucosylated include antibodies that have at leastone fucose monosaccharide in one of its N-glycans. Antibodies that arefucosylated include antibodies that have a fucose monosaccharide in eachof its N-glycans.

In some embodiments, the population of anti-TNF-alpha antibodiesdisclosed herein relates to a population wherein the level ofgalactosylation of the antibody N-glycans in the population is at least60% and the level of fucosylation of the antibodies in the population isat least 50%. In some embodiments, the population of antibodiesdisclosed herein relates to a population wherein the level ofgalactosylation of the antibody N-glycans in the population is at least50%, and the level of fucosylation of the antibody N-glycans in thepopulation is at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, up to 100%. In some embodiments, the population of antibodiesdisclosed herein relates to a population wherein the level ofgalactosylation of the antibody N-glycans in the population is at least60%, and the level of fucosylation of the antibody N-glycans in thepopulation is at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, up to 100%. In some embodiments, the population of antibodiesdisclosed herein relates to a population wherein the level ofgalactosylation of the antibody N-glycans in the population is at least70%, and the level of fucosylation of the antibody N-glycans in thepopulation is at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, up to 100%. In some embodiments, the population of antibodiesdisclosed herein relates to a population wherein the level ofgalactosylation of the antibody N-glycans in the population is at least80%, and the level of fucosylation of the antibody N-glycans in thepopulation is at least 60%, at least 70%, at least 80%, at least 90%, upto 100%. In some embodiments, the population of antibodies disclosedherein relates to a population wherein the level of galactosylation ofthe antibody N-glycans in the population is at least 90%, and the levelof fucosylation of the antibody N-glycans in the population is at least60%, at least 70%, at least 80%, at least 90%, up to 100%. In someembodiments, the population of antibodies disclosed herein relates to apopulation wherein the level of galactosylation of the antibodyN-glycans in the population is up to 100% and the level of fucosylationof the antibody N-glycans in the population is at least 60%, at least70%, at least 80%, at least 90%, up to 100%.

In one aspect, the disclosure relates to a composition comprising apopulation of anti-TNF-alpha antibodies with a specific ratio of thepercentage of antibody N-glycans in the population that aregalactosylated at the Fc-gamma-glycosylation site to the percentage ofantibody N-glycans in the population that are fucosylated at theFc-gamma-glycosylation site. In some embodiments, the disclosure relatesto a composition comprising a population of antibodies wherein the ratioof the level of galactosylation of the antibody N-glycans in thepopulation to the level of fucosylation of the antibody N-glycans in thepopulation is between 0.5 and 2.5, between 0.6 and 2.0, between 0.7 and1.8, between 0.8 and 1.6, or between 1.0 and 1.4. In some embodiments,the disclosure relates to a composition comprising a population ofantibodies wherein the ratio of the level of galactosylation of theantibody N-glycans in the population to the level of fucosylation of theantibody N-glycans in the population is between 1.0 and 1.4, for example1.2.

In some embodiments, the antibodies and populations of antibodiesdisclosed herein are highly sialylated. In some embodiments, sialylationrefers to 2,6 alpha sialylation on the terminal galactose residues ofthe Fc glycans. In some embodiments, in a population of highlysialylated antibodies at least 50% of the terminal galactose moietiesare sialylated. In some embodiments, in a population of highlysialylated antibodies at least 50%, at least 60%, at least 70%, at least80%, at least 90% and up to 100% of the terminal galactose moieties aresialylated. In some embodiments, the disclosure provides populations ofantibodies that are at least 60% galactosylated and in which at least50%, at least 60%, at least 70%, at least 80%, at least 90% and up to100% of the terminal galactose moieties are sialylated. In someembodiments, the disclosure provides populations of antibodies that areat least 70% galactosylated and in which at least 50%, at least 60%, atleast 70%, at least 80%, at least 90% and up to 100% of the terminalgalactose moieties are sialylated. In some embodiments, the disclosureprovides populations of antibodies that are at least 80% galactosylatedand in which at least 50%, at least 60%, at least 70%, at least 80%, atleast 90% and up to 100% of the terminal galactose moieties aresialylated. In some embodiments, the disclosure provides populations ofantibodies that are at least 90% galactosylated and in which at least50%, at least 60%, at least 70%, at least 80%, at least 90% and up to100% of the terminal galactose moieties are sialylated.

In some embodiments, the population of anti-TNF-alpha antibodies with ahigh level of galactosylation is produced in mammary epithelial cells ofa non-human mammal. In some embodiments, the population ofanti-TNF-alpha antibodies is produced in a transgenic non-human mammal.In some embodiments, the non-human mammal is a goat, sheep, bison,camel, cow, pig, rabbit, buffalo, horse, rat, mouse or llama. In someembodiments, the non-human mammal is a goat.

In some embodiments, the population of anti-TNF-alpha antibodies with ahigh level of galactosylation is produced in cells other than mammaryepithelial cells of a non-human mammal. In some embodiments, thepopulation of anti-TNF-alpha antibodies is modified after production incells other than mammary epithelial cells of a non-human mammal toincrease the number of galactose groups in the population of antibodies(e.g., through the action of enzymes such as transferases).

In one aspect, the disclosure provides compositions comprisingpopulations of anti-TNF-alpha antibodies with a high level ofgalactosylation. In some embodiments, the composition comprisinganti-TNF-alpha antibodies with a high level of galactosylation furthercomprises milk. In some embodiments, the composition comprisinganti-TNF-alpha antibodies with a high level of galactosylation furthercomprises a pharmaceutically-acceptable carrier.

Production of Populations of Antibodies

In one aspect, the disclosure provides compositions comprisingpopulations of anti-TNF-alpha antibodies with high levels ofgalactosylation (e.g., at least 70%), wherein the population ofantibodies is produced in mammary epithelial cells of a non-humanmammal, and wherein the population of antibodies has an increased levelof galactosylation when compared to the population of antibodies notproduced in mammary gland epithelial cells. In some embodiments, thepopulation of antibodies not produced in mammary gland epithelial cellsis produced in cell culture. As used herein, antibodies “produced incell culture” when compared to antibodies produced in mammary epithelialcells, refers to antibodies produced in standard production cell lines(e.g., CHO cells) but excluding mammary epithelial cells. In someembodiments, the level of galactosylation of the antibodies not producedin mammary gland epithelial cells is 90% or lower, 80% or lower, 70% orlower, 60% or lower, 50% or lower, 40% or lower, 30% or lower, 20% orlower, 10% or lower when compared to the level of galactosylation of theantibodies produced in mammary epithelial cells of a non-human mammal.In some embodiments, the level of galactosylation of the antibodies notproduced in mammary gland epithelial cells is 50% or lower when comparedto the level of galactosylation of the antibodies produced in mammaryepithelial cells of a non-human mammal. In some embodiments, the levelof galactosylation of the antibodies not produced in mammary glandepithelial cells is 30% or lower when compared to the level ofgalactosylation of the antibodies produced in mammary epithelial cellsof a non-human mammal. In some embodiments, the level of galactosylationof the antibodies not produced in mammary gland epithelial cells is 10%or lower when compared to the level of galactosylation of the antibodiesproduced in mammary epithelial cells of a non-human mammal.

In one aspect, the disclosure provides compositions comprisingpopulations of anti-TNF-alpha antibodies with high levels offucosylation (e.g., at least 60%), wherein the population of antibodiesis produced in mammary epithelial cells of a non-human mammal, andwherein the population of antibodies has an increased level offucosylation when compared to the population of antibodies not producedin mammary gland epithelial cells. In some embodiments, the populationof antibodies not produced in mammary gland epithelial cells is producedin cell culture. In some embodiments, the level of fucosylation of theantibodies not produced in mammary gland epithelial cells is 90% orlower, 80% or lower, 70% or lower, 60% or lower, 50% or lower, 40% orlower, 30% or lower, 20% or lower, 10% or lower when compared to thelevel of fucosylation of the antibodies produced in mammary epithelialcells of a non-human mammal. In some embodiments, the level offucosylation of the antibodies not produced in mammary gland epithelialcells is 50% or lower when compared to the level of fucosylation of theantibodies produced in mammary epithelial cells of a non-human mammal.In some embodiments, the level of fucosylation of the antibodies notproduced in mammary gland epithelial cells is 30% or lower when comparedto the level of fucosylation of the antibodies produced in mammaryepithelial cells of a non-human mammal. In some embodiments, the levelof fucosylation of the antibodies not produced in mammary glandepithelial cells is 10% or lower when compared to the level offucosylation of the antibodies produced in mammary epithelial cells of anon-human mammal.

In one aspect, the disclosure provides compositions comprisingpopulations of anti-TNF-alpha antibodies with high levels ofgalactosylation and fucosylation, wherein the population of antibodiesis produced in mammary epithelial cells of a non-human mammal, andwherein the population of antibodies has an increased level ofgalactosylation and fucosylation when compared to the population ofantibodies not produced in mammary gland epithelial cells.

Antibodies

In some embodiments, the term “antibody” refers to a glycoproteincomprising at least two heavy (H) chains and two light (L) chains. Eachheavy chain is comprised of a heavy chain variable region (abbreviatedherein as HCVR or VH) and a heavy chain constant region. The heavy chainconstant region is comprised of three domains, CH1, CH2 and CH3. Eachlight chain is comprised of a light chain variable region (abbreviatedherein as LCVR or VL) and a light chain constant region. The light chainconstant region is comprised of one domain, CL. The VH and VL regionscan be further subdivided into regions of hypervariability, termedcomplementarity determining regions (CDR), interspersed with regionsthat are more conserved, termed framework regions (FR). Each VH and VLis composed of three CDRs and four FRs, arranged from amino-terminus tocarboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3,CDR3, FR4. The variable regions of the heavy and light chains contain abinding domain that interacts with an antigen. In some embodiments, theantigen is TNF-alpha, either in soluble form, in transmembrane form, orboth. The constant regions of the antibodies may mediate the binding ofthe immunoglobulin to host tissues or factors, including various cellsof the immune system (e.g., effector cells) and the first component(C1q) of the classical complement system. Formation of a maturefunctional antibody molecule can be accomplished when two proteins areexpressed in stoichiometric quantities and self-assemble with the properconfiguration.

The term “antibodies” is also meant to encompass antigen-bindingfragments thereof. Methods for making antibodies and antigen-bindingfragments are well known in the art (see, e.g., Sambrook et al,“Molecular Cloning: A Laboratory Manual” (2nd Ed.), Cold Spring HarborLaboratory Press (1989); Lewin, “Genes IV”, Oxford University Press, NewYork, (1990), and Roitt et al., “Immunology” (2nd Ed.), Gower MedicalPublishing, London, N.Y. (1989), WO2006/040153, WO2006/122786, andWO2003/002609). As used herein, an “antigen-binding fragment” of anantibody refers to one or more portions of an antibody that retain theability to specifically bind to an antigen, e.g., TNF-alpha. It has beenshown that the antigen-binding function of an antibody can be performedby fragments of a full-length antibody. Examples of binding fragmentsencompassed within the term “antigen-binding fragment” of an antibodyinclude (i) a Fab fragment, a monovalent fragment consisting of the VL,VH, CL and CH1 domains; (ii) a F(ab′)2 fragment, a bivalent fragmentcomprising two Fab fragments linked by a disulfide bridge at the hingeregion; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) aFv fragment consisting of the VL and VH domains of a single arm of anantibody, (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546)which consists of a VH domain; and (vi) an isolated complementaritydetermining region (CDR). Furthermore, although the two domains of theFv fragment, V and VH, are coded for by separate genes, they can bejoined, using recombinant methods, by a synthetic linker that enablesthem to be made as a single protein chain in which the VL and VH regionspair to form monovalent molecules (known as single chain Fv (scFv); seee.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988)Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single chain antibodiesare also intended to be encompassed within the term “antigen-bindingportion” of an antibody. These antibody fragments are obtained usingconventional procedures, such as proteolytic fragmentation procedures,as described in J. Goding, Monoclonal Antibodies: Principles andPractice, pp 98-118 (N.Y. Academic Press 1983), which is herebyincorporated by reference as well as by other techniques known to thosewith skill in the art. The fragments are screened for utility in thesame manner as are intact antibodies.

In some embodiments the antibodies are of the isotype IgG, IgA or IgD.In further embodiments, the antibodies are selected from the groupconsisting of IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgAsec, IgD, IgEor has immunoglobulin constant and/or variable domain of IgG1, IgG2,IgG3, IgG4, IgM, IgA1, IgA2, IgAsec, IgD or IgE. In other embodiments,the antibodies are bispecific or multispecific antibodies. According toan alternative embodiment, the antibodies of the present disclosure canbe modified to be in the form of a bispecific antibody, or amultispecific antibody. The term “bispecific antibody” is intended toinclude any agent, e.g., a protein, peptide, or protein or peptidecomplex, which has two different binding specificities which bind to, orinteract with (a) a cell surface antigen and (b) an Fc receptor on thesurface of an effector cell. The term “multispecific antibody” isintended to include any agent, e.g., a protein, peptide, or protein orpeptide complex, which has more than two different binding specificitieswhich bind to, or interact with (a) a cell surface antigen, (b) an Fcreceptor on the surface of an effector cell, and (c) at least one othercomponent. Accordingly, the disclosure includes, but is not limited to,bispecific, trispecific, tetraspecific, and other multispecificantibodies which are directed to cell surface antigens, and to Fcreceptors on effector cells. The term “bispecific antibodies” furtherincludes diabodies. Diabodies are bivalent, bispecific antibodies inwhich the VH and VL domains are expressed on a single polypeptide chain,but using a linker that is too short to allow for pairing between thetwo domains on the same chain, thereby forcing the domains to pair withcomplementary domains of another chain and creating two antigen-bindingsites (see e.g., Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA90:6444-6448; Poijak, R. J., et al. (1994) Structure 2:1121-1123).

The term “antibodies” also encompasses different types of antibodies,e.g., recombinant antibodies, monoclonal antibodies, humanizedantibodies or chimeric antibodies, or a mixture of these.

In some embodiments, the antibodies are recombinant antibodies. The term“recombinant antibody”, as used herein, is intended to includeantibodies that are prepared, expressed, created or isolated byrecombinant means, such as antibodies isolated from an animal that istransgenic for another species' immunoglobulin genes, antibodiesexpressed using a recombinant expression vector transfected into a hostcell, antibodies isolated from a recombinant, combinatorial antibodylibrary, or antibodies prepared, expressed, created or isolated by anyother means that involves splicing of immunoglobulin gene sequences toother DNA sequences.

In yet other embodiments, the antibodies can be chimeric or humanizedantibodies. As used herein, the term “chimeric antibody” refers to anantibody that combines parts of a non-human (e.g., mouse, rat, rabbit)antibody with parts of a human antibody. As used herein, the term“humanized antibody” refers to an antibody that retains only theantigen-binding CDRs from the parent antibody in association with humanframework regions (see, Waldmann, 1991, Science 252:1657). Such chimericor humanized antibodies retaining binding specificity of the murineantibody are expected to have reduced immunogenicity when administeredin vivo for diagnostic, prophylactic or therapeutic applicationsaccording to the disclosure.

In certain embodiments, the antibodies are human antibodies. The term“human antibody”, as used herein, is intended to include antibodieshaving variable and constant regions derived from human germlineimmunoglobulin sequences. The human antibodies of the disclosure mayinclude amino acid residues not encoded by human germline immunoglobulinsequences (e.g., mutations introduced by random or site-specificmutagenesis in vitro or by somatic mutation in vivo). Human antibodiesare generated using transgenic mice carrying parts of the human immunesystem rather than the mouse system. Fully human monoclonal antibodiesalso can be prepared by immunizing mice transgenic for large portions ofhuman immunoglobulin heavy and light chain loci. See, e.g., U.S. Pat.Nos. 5,591,669, 5,598,369, 5,545,806, 5,545,807, 6,150,584, andreferences cited therein, the contents of which are incorporated hereinby reference. These animals have been genetically modified such thatthere is a functional deletion in the production of endogenous (e.g.,murine) antibodies. The animals are further modified to contain all or aportion of the human germ-line immunoglobulin gene locus such thatimmunization of these animals results in the production of fully humanantibodies to the antigen of interest. Following immunization of thesemice (e.g., XenoMouse (Abgenix), HuMAb mice (Medarex/GenPharm)),monoclonal antibodies are prepared according to standard hybridomatechnology. These monoclonal antibodies have human immunoglobulin aminoacid sequences and therefore will not provoke human anti-mouse antibody(HAMA) responses when administered to humans. The human antibodies, likeany of the antibodies provided herein can be monoclonal antibodies.

In some embodiments, the antibody is a full-length antibody. In someembodiments the full-length antibody comprises a heavy chain and a lightchain. In some embodiments, the antibody is an anti-TNF-alpha antibody.In some embodiments, the heavy chain comprises SEQ ID NO: 1 and thelight chain comprises SEQ ID NO: 2. In some embodiments, the antibody isadalimumab.

CDC Activity

In one aspect, the compositions comprising populations of anti-TNF-alphaantibodies with high levels of galactosylation (e.g., at least 70%) havehigh complement dependent cytotoxicity (CDC) activity. In one aspect,the compositions comprising populations of anti-TNF-alpha antibodieswith high levels of galactosylation have high antibody-dependentcellular cytotoxicity (ADCC) activity. In some embodiments, thecompositions comprising populations of anti-TNF-alpha antibodies withhigh levels of galactosylation have high complement dependentcytotoxicity (CDC) activity and have high antibody-dependent cellularcytotoxicity (ADCC) activity.

In some embodiments, the population of anti-TNF-alpha antibodies withhigh levels of galactosylation has an increased level of complementdependent cytotoxicity (CDC) activity when compared to a population ofantibodies that have low levels of galactosylation. In some embodiments,the population of antibodies with high levels of galactosylation and thepopulation of antibodies that have low levels of galactosylation aredirected to the same antigen epitope. In some embodiments, thepopulation of antibodies that is highly galactosylated and thepopulation of antibodies that have low levels of galactosylation areencoded by the same nucleic acid. In some embodiments, the nucleic acidencodes the antibody adalimumab.

A population of antibodies that has low levels of galactosylation (is“low galactose”), as used herein, refers to a population of antibodieswherein the level of galactosylation of the antibodies in the populationis less than 50%, less than 40%, less than 30%, less than 20%, less than10%, down to 0%.

In some embodiments, the CDC activity of a population of antibodies withhigh levels of galactosylation is at least 1.1 times higher, at least1.2 times higher, at least 1.3 times higher, at least 1.4 times higher,at least 1.5 times higher, at least 1.6 times higher, at least 1.7 timeshigher, at least 1.8 times higher, at least 1.9 times higher, at least 2times higher, at least 3 times higher, at least 5 times higher, at least10 times higher, up to at least 100 times higher or more when comparedto a population of antibodies that have low levels of galactosylation.

In some embodiments, the population of antibodies that are highlygalactosylated are highly fucosylated (have high levels offucosylation). In some embodiments, the population of antibodies thatare highly galactosylated and highly fucosylated has an increased levelof complement dependent cytotoxicity (CDC) activity when compared to apopulation of antibodies that are low galactose and low fucose (have lowlevels of galactosylation and fucosylation). In some embodiments, thepopulation of antibodies that is highly galactosylated and highlyfucosylated and the population of antibodies that is low galactose andlow fucose are directed to the same antigen epitope. In someembodiments, the population of antibodies that is highly galactosylatedand highly fucosylated and the population of antibodies that is lowgalactose and low fucose are encoded by the same nucleic acid. In someembodiments, the nucleic acid encodes the antibody adalimumab.

A population of antibodies that are low fucose or that have low levelsof fucosylation, as used herein, refers to a population of antibodieswherein the level of fucosylation of the antibodies in the population isless than 50%, less than 40%, less than 30%, less than 20%, less than10%, down to 0%.

In some embodiments, the CDC activity of a population of antibodies thatis highly galactosylated and highly fucosylated is at least 1.1 timeshigher, at least 1.2 times higher, at least 1.3 times higher, at least1.4 times higher, at least 1.5 times higher, at least 1.6 times higher,at least 1.7 times higher, at least 1.8 times higher, at least 1.9 timeshigher, at least 2 times higher, at least 3 times higher, at least 5times higher, at least 10 times higher, up to at least 100 times higheror more when compared to a population of antibodies that is lowgalactose and low fucose.

In some embodiments, the population of antibodies that is highlygalactosylated and is produced in mammary gland epithelial cells has anincreased level of complement dependent cytotoxicity (CDC) activity whencompared to a population of antibodies that is not produced in mammarygland epithelial cells. In some embodiments, the population ofantibodies not produced in mammary gland epithelial cells is produced incell culture. In some embodiments, the population of antibodies that ishighly galactosylated produced in mammary gland epithelial cells and thepopulation of antibodies that is not produced in mammary glandepithelial cells may be encoded by the same nucleic acid. In someembodiments, the nucleic acid encodes the antibody adalimumab.

In some embodiments, the CDC activity of a population of antibodies thatis highly galactosylated and is produced in mammary gland epithelialcells is at least 1.1 times higher, at least 1.2 times higher, at least1.3 times higher, at least 1.4 times higher, at least 1.5 times higher,at least 1.6 times higher, at least 1.7 times higher, at least 1.8 timeshigher, at least 1.9 times higher, at least 2 times higher, at least 3times higher, at least 5 times higher, at least 10 times higher, up toat least 100 times higher or more when compared to a population ofantibodies that is not produced in mammary gland epithelial cells.

In one aspect, the compositions of the populations of antibodiesdisclosed herein have a high (complement dependent cytotoxicity) CDCactivity. Antibodies can act as a therapeutic through variousmechanisms, one of which is through CDC activity. Some therapeuticantibodies that bind to target cellular receptors can also bind proteinsof the complement pathway. Binding of the complement proteins results ina complement cascade (through C1-complex activation) that eventuallyresults in the formation of a “membrane attack complex” causing celllysis and death of the cell to which the therapeutic antibody is bound(See e.g., Reff M. E. Blood 1994, 83: 435).

In some embodiments a population of antibodies that has an increasedlevel of complement dependent cytotoxicity (CDC) activity, is apopulation of antibodies that induces a larger amount of cell lysis ascompared to a population of antibodies that has does not have anincreased level of complement dependent cytotoxicity (CDC) activity.Methods for determining the level of CDC are known in the art and areoften based on determining the amount of cell lysis. Commercial kits fordetermining CDC activity can be purchased for instance from Genscript(Piscataway, N.J.).

ADCC Activity

In one aspect, the population of anti-TNF-alpha antibodies with highlevels of galactosylation (e.g., at least 70%), has an increased levelof antibody-dependent cellular cytotoxicity (ADCC) activity whencompared to a population of antibodies that have low levels ofgalactosylation. In some embodiments, the disclosure providescompositions comprising populations of anti-TNF-alpha antibodies with ahigh level of galactosylation wherein the population of antibodies isproduced in mammary epithelial cells of a non-human mammal, and whereinthe population of antibodies has an increased level ofantibody-dependent cellular cytotoxicity (ADCC) activity when comparedto a population of antibodies not produced in mammary gland epithelialcells. In some embodiments, the population of antibodies not produced inmammary gland epithelial cells is produced in cell culture. In someembodiments, the population of anti-TNF-alpha antibodies with highlevels of galactosylation (e.g., at least 70%), has an increased levelof antibody-dependent cellular cytotoxicity (ADCC) activity whencompared to a population of antibodies that is aglycosylated.

In some embodiments, the population of antibodies that are highlygalactosylated has an increased level of antibody-dependent cellularcytotoxicity (ADCC) when compared to a population of antibodies that arelow galactose. In some embodiments, the ADCC activity of a population ofantibodies that is highly galactosylated is at least 1.1 times higher,1.2 times higher, 1.3 times higher, 1.4 times higher, 1.5 times higher,1.6 times higher, 1.7 times higher, 1.8 times higher, 1.9 times higher,2 times higher, 3 times higher, 5 times higher, 10 times higher, 100times higher or more when compared to a population of antibodies thatare low galactose.

In some embodiments, the population of antibodies that are highlygalactosylated and is produced in mammary gland epithelial cells has anincreased level of antibody-dependent cellular cytotoxicity (ADCC) whencompared to a population of antibodies that is not produced in mammarygland epithelial cells. In some embodiments, the ADCC activity of apopulation of antibodies that is highly galactosylated and produced inmammary gland epithelial cells is at least 1.1 times higher, 1.2 timeshigher, 1.3 times higher, 1.4 times higher, 1.5 times higher, 1.6 timeshigher, 1.7 times higher, 1.8 times higher, 1.9 times higher, 2 timeshigher, 3 times higher, 5 times higher, 10 times higher, 100 timeshigher or more when compared to a population of antibodies that is notproduced in mammary gland epithelial cells.

In some embodiments, the population of antibodies that is highlygalactosylated and is produced in mammary gland epithelial cells has anincreased level of antibody-dependent cellular cytotoxicity (ADCC) whencompared to a population of antibodies that is not produced in mammarygland epithelial cells. In some embodiments, the population ofantibodies not produced in mammary gland epithelial cells is produced incell culture.

In one aspect, the compositions of the populations of antibodiesdisclosed herein have a high ADCC activity. Antibodies can act as atherapeutic through various mechanisms, one of which is through ADCCactivity. Therapeutic antibodies that bind to cellular receptors on atarget cell, and that include the Fc glycosylation site can also bindthe Fc-receptor resulting in the anchoring of cells expressing theFc-receptor to the target cell. The affinity of binding of the Fcregions of antibodies generally is dependent on the nature of theglycosylation of the Fc glycosylation site. The Fc receptor is found ona number of immune cells including natural killer cells, macrophages,neutrophils, and mast cells. Binding to the Fc receptor results in theimmune cells inducing cytokines (such as IL-2) and phagocytosis to killthe target cell. In some embodiments, a population of antibodies thathas an increased level of antibody-dependent cellular cytotoxicity(ADCC) activity is a population of antibodies that shows increasedbinding to cells expressing CD16 as compared to a population ofantibodies that does not have an increased level of antibody-dependentcellular cytotoxicity (ADCC) activity. In some embodiments a populationof antibodies that has an increased level of antibody-dependent cellularcytotoxicity (ADCC) activity is a population of antibodies that showsincreased induction of IL-2 production (e.g., in natural killer cells)as compared to a population of antibodies that has does not have anincreased level of antibody-dependent cellular cytotoxicity (ADCC)activity. Commercial kits for determining ADCC activity can be purchasedfor instance from Genscript (Piscataway, N.J.) and Promega (Madison,Wis.). In some embodiments, determining ADCC activity is performed byevaluating the ability to bind CD16.

Anti-TNF-Alpha Activity

In one aspect, the population of anti-TNF-alpha antibodies with highlevels of galactosylation (e.g., at least 70%) has an increased abilityto suppress TNF-alpha activity in a subject when compared to apopulation of antibodies that have low levels of galactosylation. Insome embodiments, the disclosure provides compositions comprisingpopulations of anti-TNF-alpha antibodies with high levels ofgalactosylation, wherein the population of antibodies is produced inmammary epithelial cells of a non-human mammal, and wherein thepopulation of antibodies has an increased ability to suppress TNF-alphaactivity in a subject when compared to a population of antibodies notproduced in mammary gland epithelial cells.

In one aspect, the disclosure provides compositions comprisingpopulations of anti-TNF-alpha antibodies with high levels ofgalactosylation, wherein the population of antibodies is produced inmammary epithelial cells of a non-human mammal, and wherein thepopulation of antibodies has an increased ability to bind solubleTNF-alpha when compared to a population of antibodies not produced inmammary gland epithelial cells.

In one aspect, the disclosure provides compositions comprisingpopulations of anti-TNF-alpha antibodies with high levels ofgalactosylation, wherein the population of antibodies is produced inmammary epithelial cells of a non-human mammal, and wherein thepopulation of antibodies has an increased ability to bind transmembraneTNF-alpha when compared to a population of antibodies not produced inmammary gland epithelial cells.

In some embodiments, the population of antibodies that are highlygalactosylated has an increased ability to suppress TNF-alpha activity,bind soluble TNF-alpha and/or bind transmembrane TNF-alpha when comparedto a population of antibodies that are low galactose. In someembodiments, the increased ability to suppress TNF-alpha activity, bindsoluble TNF-alpha and/or bind transmembrane TNF-alpha of a population ofantibodies that is highly galactosylated is at least 1.1 times higher,1.2 times higher, 1.3 times higher, 1.4 times higher, 1.5 times higher,1.6 times higher, 1.7 times higher, 1.8 times higher, 1.9 times higher,2 times higher, 3 times higher, 5 times higher, 10 times higher, 100times higher or more when compared to a population of antibodies thatare low galactose.

In some embodiments, the population of antibodies that are highlygalactosylated and is produced in mammary gland epithelial cells has anincreased ability to suppress TNF-alpha activity, bind soluble TNF-alphaand/or bind transmembrane TNF-alpha when compared to a population ofantibodies that is not produced in mammary gland epithelial cells. Insome embodiments, the increased ability to suppress TNF-alpha activity,bind soluble TNF-alpha and/or bind transmembrane TNF-alpha of apopulation of antibodies that is highly galactosylated and produced inmammary gland epithelial cells is at least 1.1 times higher, 1.2 timeshigher, 1.3 times higher, 1.4 times higher, 1.5 times higher, 1.6 timeshigher, 1.7 times higher, 1.8 times higher, 1.9 times higher, 2 timeshigher, 3 times higher, 5 times higher, 10 times higher, 100 timeshigher or more when compared to a population of antibodies that is notproduced in mammary gland epithelial cells.

In some embodiments, the population of antibodies that is highlygalactosylated and is produced in mammary gland epithelial cells hasincreased ability to suppress TNF-alpha activity, bind soluble TNF-alphaand/or bind transmembrane TNF-alpha when compared to a population ofantibodies that is not produced in mammary gland epithelial cells. Insome embodiments, the population of antibodies not produced in mammarygland epithelial cells is produced in cell culture.

In some embodiments, the populations of anti-TNF-alpha antibodiesproduced in mammary gland epithelial cells are superior to non-mammarygland epithelial cells produced antibodies in suppressing TNF-alphaactivity in a subject. Determining the level of TNF-alpha activity in asubject can be evaluated for instance, by administering the populationof antibodies to a subject suffering from a disease characterized byincreased TNF-alpha activity (e.g., rheumatoid arthritis) or anestablished model for such a disease. (See e.g., Horiuchi et al.,Rheumatology et al. 49:1215).

In some embodiments, the populations of anti-TNF-alpha antibodiesproduced in mammary gland epithelial cells are superior to non-mammarygland epithelial cells produced antibodies in binding soluble TNF-alpha.In some embodiments, the populations of anti-TNF-alpha antibodiesproduced in mammary gland epithelial cells are superior to non-mammarygland epithelial cells produced antibodies in binding transmembraneTNF-alpha. Assays for determining the level of binding to solubleTNF-alpha or transmembrane TNF-alpha are well-established (See e.g.,Horiuchi et al., Rheumatology et al. 49:1215).

Non-Human Mammary Gland Epithelial Cells and Transgenic Animals

In one aspect, the disclosure provides mammary gland epithelial cellsthat produce highly galactosylated anti-TNF-alpha antibodies orpopulations of anti-TNF-alpha antibodies with a high level ofgalactosylation.

In one aspect, the disclosure provides a transgenic non-human mammalthat produces highly galactosylated anti-TNF-alpha antibody orpopulations of anti-TNF-alpha antibodies with a high level ofgalactosylation

In one aspect, the disclosure relates to mammalian mammary epithelialcells that produce glycosylated antibodies. Methods are provided hereinfor producing glycosylated antibodies in mammalian mammary epithelialcells. This can be accomplished in cell culture by culturing mammaryepithelial cell (in vitro or ex vivo). This can also be accomplished ina transgenic animal (in vivo).

In some embodiments, the mammalian mammary gland epithelial cells are ina transgenic animal. In some embodiments, the mammalian mammary glandepithelial cells have been engineered to express recombinant antibodiesin the milk of a transgenic animal, such as a mouse or goat. Toaccomplish this, the expression of the gene(s) encoding the recombinantprotein can be, for example, under the control of the goat β-caseinregulatory elements. Expression of recombinant proteins, e.g.,antibodies, in both mice and goat milk has been established previously(see, e.g., US Patent Application US-2008-0118501-A1). In someembodiments, the expression is optimized for individual mammary ductepithelial cells that produce milk proteins.

Transgenic animals capable of producing recombinant antibodies can begenerated according to methods known in the art (see, e.g., U.S. Pat.No. 5,945,577 and US Patent Application US-2008-0118501-A1). Animalssuitable for transgenic expression, include, but are not limited togoat, sheep, bison, camel, cow, pig, rabbit, buffalo, horse, rat, mouseor llama. Suitable animals also include bovine, caprine, ovine andporcine, which relate to various species of cows, goats, sheep and pigs(or swine), respectively. Suitable animals also include ungulates. Asused herein, “ungulate” is of or relating to a hoofed typicallyherbivorous quadruped mammal, including, without limitation, sheep,swine, goats, cattle and horses. Suitable animals also include dairyanimals, such as goats and cattle, or mice. In some embodiments, theanimal suitable for transgenic expression is a goat.

In one embodiment, transgenic animals are generated by generation ofprimary cells comprising a construct of interest followed by nucleartransfer of primary cell nucleus into enucleated oocytes. Primary cellscomprising a construct of interest are produced by injecting ortransfecting primary cells with a single construct comprising the codingsequence of an antibody of interest, e.g., the heavy and light chains ofadalimumab, or by co-transfecting or co-injecting primary cells withseparate constructs comprising the coding sequences of the heavy andlight chains of an antibody, e.g., adalimumab. These cells are thenexpanded and characterized to assess transgene copy number, transgenestructural integrity and chromosomal integration site. Cells withdesired transgene copy number, transgene structural integrity andchromosomal integration site are then used for nuclear transfer toproduce transgenic animals. As used herein, “nuclear transfer” refers toa method of cloning wherein the nucleus from a donor cell istransplanted into an enucleated oocyte.

Coding sequences for antibodies to be expressed in mammalian mammaryepithelial cells can be obtained by screening libraries of genomicmaterial or reverse-translated messenger RNA derived from the animal ofchoice (such as humans, cattle or mice), from sequence databases such asNCBI, Genbank, or by obtaining the sequences of antibodies using methodsknown in the art, e.g. peptide mapping. The sequences can be cloned intoan appropriate plasmid vector and amplified in a suitable host organism,like E. coli. As used herein, a “vector” may be any of a number ofnucleic acids into which a desired sequence may be inserted byrestriction and ligation for transport between different geneticenvironments or for expression in a host cell. Vectors are typicallycomposed of DNA although RNA vectors are also available. Vectorsinclude, but are not limited to, plasmids and phagemids. A cloningvector is one which is able to replicate in a host cell, and which isfurther characterized by one or more endonuclease restriction sites atwhich the vector may be cut in a determinable fashion and into which adesired DNA sequence may be ligated such that the new recombinant vectorretains its ability to replicate in the host cell. An expression vectoris one into which a desired DNA sequence may be inserted by restrictionand ligation such that it is operably joined to regulatory sequences andmay be expressed as an RNA transcript. Vectors may further contain oneor more marker sequences suitable for use in the identification of cellswhich have or have not been transformed or transfected with the vector.Markers include, for example, genes encoding proteins which increase ordecrease either resistance or sensitivity to antibiotics or othercompounds, genes which encode enzymes whose activities are detectable bystandard assays known in the art (e.g., β-galactosidase or alkalinephosphatase), and genes which visibly affect the phenotype oftransformed or transfected cells, hosts, colonies or plaques.

The coding sequence of antibodies or the heavy and light chains ofantibodies of interest can be operatively linked to a control sequencewhich enables the coding sequence to be expressed in the milk of atransgenic non-human mammal. After amplification of the vector, the DNAconstruct can be excised, purified from the remains of the vector andintroduced into expression vectors that can be used to producetransgenic animals. The transgenic animals will have the desiredtransgenic protein integrated into their genome.

A DNA sequence which is suitable for directing production to the milk oftransgenic animals can carry a 5′-promoter region derived from anaturally-derived milk protein. This promoter is consequently under thecontrol of hormonal and tissue-specific factors and is most active inlactating mammary tissue. In some embodiments the promoter used is amilk-specific promoter. As used herein, a “milk-specific promoter” is apromoter that naturally directs expression of a gene in a cell thatsecretes a protein into milk (e.g., a mammary epithelial cell) andincludes, for example, the casein promoters, e.g., α-casein promoter(e.g., alpha S-1 casein promoter and alpha S2-casein promoter), β-caseinpromoter (e.g., the goat beta casein gene promoter (DiTullio,BIOTECHNOLOGY 10:74-77, 1992), γ-casein promoter, κ-casein promoter,whey acidic protein (WAP) promoter (Gorton et al., BIOTECHNOLOGY 5:1183-1187, 1987), β-lactoglobulin promoter (Clark et al., BIOTECHNOLOGY7: 487-492, 1989) and α-lactalbumin promoter (Soulier et al., FEBSLETTS. 297:13, 1992). Also included in this definition are promotersthat are specifically activated in mammary tissue, such as, for example,the long terminal repeat (LTR) promoter of the mouse mammary tumor virus(MMTV). In some embodiments the promoter is a caprine beta caseinpromoter.

The promoter can be operably linked to a DNA sequence directing theproduction of a protein leader sequence which directs the secretion ofthe transgenic protein across the mammary epithelium into the milk. Asused herein, a coding sequence and regulatory sequences (e.g., apromoter) are said to be “operably joined” or “operably linked” whenthey are linked in such a way as to place the expression ortranscription of the coding sequence under the influence or control ofthe regulatory sequences. As used herein, a “leader sequence” or “signalsequence” is a nucleic acid sequence that encodes a protein secretorysignal, and, when operably linked to a downstream nucleic acid moleculeencoding a transgenic protein directs secretion. The leader sequence maybe the native human leader sequence, an artificially-derived leader, ormay be obtained from the same gene as the promoter used to directtranscription of the transgene coding sequence, or from another proteinthat is normally secreted from a cell, such as a mammalian mammaryepithelial cell. In some embodiments a 3′-sequence, which can be derivedfrom a naturally secreted milk protein, can be added to improvestability of mRNA.

In some embodiments, to produce primary cell lines containing aconstruct (e.g., encoding an adalimumab antibody) for use in producingtransgenic goats by nuclear transfer, the heavy and light chainconstructs can be transfected into primary goat skin epithelial cells,which are expanded and fully characterized to assess transgene copynumber, transgene structural integrity and chromosomal integration site.As used herein, “nuclear transfer” refers to a method of cloning whereinthe nucleus from a donor cell is transplanted into an enucleated oocyte.

Cloning will result in a multiplicity of transgenic animals—each capableof producing an antibody or other gene construct of interest. Theproduction methods include the use of the cloned animals and theoffspring of those animals. Cloning also encompasses the nucleartransfer of fetuses, nuclear transfer, tissue and organ transplantationand the creation of chimeric offspring. One step of the cloning processcomprises transferring the genome of a cell, e.g., a primary cell thatcontains the transgene of interest into an enucleated oocyte. As usedherein, “transgene” refers to any piece of a nucleic acid molecule thatis inserted by artifice into a cell, or an ancestor thereof, and becomespart of the genome of an animal which develops from that cell. Such atransgene may include a gene which is partly or entirely exogenous(i.e., foreign) to the transgenic animal, or may represent a gene havingidentity to an endogenous gene of the animal. Suitable mammalian sourcesfor oocytes include goats, sheep, cows, pigs, rabbits, guinea pigs,mice, hamsters, rats, non-human primates, etc. Preferably, oocytes areobtained from ungulates, and most preferably goats or cattle. Methodsfor isolation of oocytes are well known in the art. Essentially, theprocess comprises isolating oocytes from the ovaries or reproductivetract of a mammal, e.g., a goat. A readily available source of ungulateoocytes is from hormonally-induced female animals. For the successfuluse of techniques such as genetic engineering, nuclear transfer andcloning, oocytes may preferably be matured in vivo before these cellsmay be used as recipient cells for nuclear transfer, and before theywere fertilized by the sperm cell to develop into an embryo. MetaphaseII stage oocytes, which have been matured in vivo, have beensuccessfully used in nuclear transfer techniques. Essentially, maturemetaphase II oocytes are collected surgically from either non-superovulated or super ovulated animals several hours past the onset ofestrus or past the injection of human chorionic gonadotropin (hCG) orsimilar hormone.

In some embodiments, the transgenic animals (e.g., goats) and mammaryepithelial cells are generated through microinjection. Microinjection ingoats is described for instance in U.S. Pat. No. 7,928,064. Briefly,fertilized goat eggs are collected from the PBS oviductal flushings on astereomicroscope, and washed in medium containing 10% fetal bovine serum(FBS). In cases where the pronuclei were visible, the embryos can beimmediately microinjected. If pronuclei are not visible, the embryos canbe placed media for short term culture until the pronuclei becamevisible (Selgrath, et al., Theriogenology, 1990. p. 1195-1205). One-cellgoat embryos are placed in a microdrop of medium under oil on a glassdepression slide. Fertilized eggs having two visible pronuclei and canbe immobilized on a flame-polished holding micropipet on an uprightmicroscope with a fixed stage. A pronucleus can be microinjected withthe appropriate antibody encoding construct in injection buffer using afine glass microneedle (Selgrath, et al., Theriogenology, 1990. p.1195-1205). After microinjection, surviving embryos are placed in aculture and incubated until the recipient animals are prepared forembryo transfer (Selgrath, et al., Theriogenology, 1990. p. 1195-1205).

Thus, in one aspect the disclosure provides mammary gland epithelialcells that produce the antibodies or populations of antibodies disclosedherein. In some embodiments, the antibody comprises a nucleic acidcomprising SEQ ID NO: 3 and a nucleic acid comprising SEQ ID NO: 4. Insome embodiments, the nucleic acid comprising SEQ ID NO: 3 and thenucleic acid comprising SEQ ID NO: 4 are connected. “Connected” is usedherein to mean the nucleic acids are physically linked, e.g., within thesame vector or within approximately the same genomic location. In someembodiments, the mammary epithelial cells above are in a transgenicnon-human mammal. In some embodiments, the transgenic non-human mammalis a goat.

A nucleic acid sequence encoding the heavy chain of adalimumab isprovided in SEQ ID NO:3:

ATGGAATTCGGCCTGAGCTGGCTGTTCCTGGTGGCCATCCTGAAGGGCGTGCAGTGCGAGGTGCAGCTGGTGGAGTCTGGCGGAGGACTGGTGCAGCCCGGCAGAAGCCTGAGACTGAGCTGCGCCGCCAGCGGCTTCACCTTCGACGACTACGCCATGCACTGGGTCCGCCAGGCCCCTGGAAAGGGCCTGGAATGGGTGTCCGCCATCACCTGGAACAGCGGCCACATCGACTACGCCGACAGCGTGGAGGGCAGGTTCACCATCAGCAGGGACAACGCCAAGAACAGCCTGTACCTGCAGATGAACAGCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAAGGTGTCCTACCTGAGCACCGCCAGCAGCCTGGATTACTGGGGCCAGGGCACCCTGGTGACCGTGTCCAGCGCCAGCACCAAGGGCCCTAGCGTGTTCCCTCTGGCCCCCAGCAGCAAGTCTACCTCTGGCGGCACAGCCGCTCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAGCCCGTGACAGTGTCCTGGAACTCTGGCGCCCTGACCAGCGGCGTGCACACATTCCCTGCCGTGCTGCAGAGCAGCGGCCTGTACAGCCTGAGCAGCGTGGTGACAGTGCCTAGCAGCTCTCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGAGCCCAAGAGCTGCGACAAGACCCACACCTGTCCCCCTTGTCCTGCCCCTGAGCTGCTGGGCGGACCCAGCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCAGCAGGACCCCCGAAGTGACCTGCGTGGTGGTGGACGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAGGTGCACAATGCCAAGACCAAGCCCAGAGAGGAACAGTACAACAGCACCTACAGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTCTCCAACAAGGCCCTGCCTGCCCCCATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCCAGAGAACCCCAGGTGTACACCCTGCCCCCTAGCAGGGACGAGCTGACCAAGAACCAGGTGTCCCTGACCTGTCTGGTGAAGGGCTTCTACCCCAGCGATATCGCCGTGGAGTGGGAGTCTAACGGCCAGCCTGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACTCCAAACTGACCGTGGACAAGAGCAGATGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGAGCCTGAG CCCCGGCAAGTAATGA

A nucleic acid sequence encoding the light chain of adalimumab isprovided in SEQ ID NO:4:

ATGGACATGAGAGTGCCCGCTCAGCTGCTGGGACTGCTGCTGCTGTGGCTGAGAGGCGCCAGATGCGACATCCAGATGACCCAGAGCCCTTCTAGCCTGAGCGCCAGCGTGGGCGACAGAGTGACCATCACCTGTAGGGCCAGCCAGGGCATCAGGAACTACCTGGCCTGGTATCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACGCCGCCAGCACCCTGCAGAGCGGCGTGCCCAGCAGATTCAGCGGCAGCGGCTCCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCTGAGGACGTGGCCACCTACTACTGCCAGAGGTACAACAGGGCCCCCTACACCTTCGGACAGGGCACCAAGGTGGAGATCAAGAGGACCGTGGCCGCTCCCAGCGTGTTCATCTTCCCACCCAGCGACGAGCAGCTGAAGTCTGGCACCGCCTCCGTGGTCTGCCTGCTGAACAACTTCTACCCCCGCGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGTCCGGCAACAGCCAGGAAAGCGTCACCGAGCAGGACAGCAAGGACTCCACCTACTCCCTGTCCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAAGTGACCCACCAGGGCCTGAGCAGCCCTGTGACCAAGAGCTTCAACAGGG GCGAGTGCTAATGA

In another aspect the disclosure provides a method for the production ofa transgenic antibody, and populations thereof, the process comprisingexpressing in the milk of a transgenic non-human mammal a transgenicantibody encoded by a nucleic acid construct. In some embodiments, themethod for producing the antibodies of the disclosure comprises:

(a) transfecting non-human mammalian cells with a transgene DNAconstruct encoding an anti-TNF-alpha antibody;

(b) selecting cells in which said anti-TNF-alpha transgene DNA constructhas been inserted into the genome of the cells; and

(c) performing a first nuclear transfer procedure to generate anon-human transgenic mammal heterozygous for the anti-TNF-alpha antibodyand that can express it in its milk.

In some embodiments, the anti-TNF-alpha antibody is adalimumab.

In some embodiments, the transgene DNA construct comprises SEQ ID NO:3and/or SEQ ID NO:4. In some embodiments, the non-human transgenic mammalis a goat.

In another aspect, the disclosure provides a method of:

(a) providing a non-human transgenic mammal engineered to express ananti-TNF-alpha antibody,

(b) expressing the anti-TNF-alpha antibody in the milk of the non-humantransgenic mammal; and

(c) isolating the anti-TNF-alpha antibody expressed in the milk.

In some embodiments, the anti-TNF-alpha antibody comprises a heavy chaincomprising SEQ ID NO:1 and a light chain comprising SEQ ID NO:2. In someembodiments, the anti-TNF-alpha antibody is adalimumab.

One of the tools used to predict the quantity and quality of therecombinant protein expressed in the mammary gland is through theinduction of lactation (Ebert K M, 1994). Induced lactation allows forthe expression and analysis of protein from the early stage oftransgenic production rather than from the first natural lactationresulting from pregnancy, which is at least a year later. Induction oflactation can be done either hormonally or manually.

In some embodiments, the compositions of glycosylated antibodiesprovided herein further comprise milk. In some embodiments, the methodsprovided herein include a step of isolating a population of antibodiesfrom the milk of a transgenic animal. Methods for isolating antibodiesfrom the milk of transgenic animal are known in the art and aredescribed for instance in Pollock et al., Journal of ImmunologicalMethods, Volume 231, Issues 1-2, 10 Dec. 1999, Pages 147-157. In someembodiments, the methods provided herein include a step of purifyingglycosylated antibodies with a desired amount of galactosylation.

Methods of Treatment, Pharmaceutical Compositions, Dosage, andAdministration

In one aspect, the disclosure provides methods comprising administeringhighly galactosylated antibodies, compositions of highly galactosylatedantibodies, populations of antibodies with a high level ofgalactosylated antibodies or compositions comprising populations ofantibodies with a high level of galactosylated antibodies, to a subjectin need thereof. In some embodiment, the subject has an inflammatorydisorder or autoimmune disorder. In some embodiment, the inflammatorydisorder or autoimmune disorder is rheumatoid arthritis, psoriasis,Crohn's disease, juvenile idiopathic arthritis, ankylozing spondylitis,ulcerative colitis, chronic inflammation, hepatitis, Behcet's disease,Wegener's granulomatosis, or sarcoidosis.

In one aspect, the disclosure provides methods for administering any oneof antibodies or compositions described herein to a subject in needthereof. In some embodiments, the subject has an immune disorder ordisorder associated with inflammation. Immune disorders and disordersassociated with inflammation include but are not limited, to adultrespiratory distress syndrome, arteriosclerosis, asthma,atherosclerosis, cholecystitis, cirrhosis, Crohn's disease, diabetesmellitus, emphysema, hypereosinophilia, inflammation, irritable bowelsyndrome, multiple sclerosis, myasthenia gravis, myocardial orpericardial inflammation, osteoarthritis, osteoporosis, pancreatitis,rheumatoid arthritis, scleroderma, colitis, systemic lupuserythematosus, lupus nephritis, diabetes mellitus, inflammatory boweldisease, celiac disease, an autoimmune thyroid disease, Addison'sdisease, Sjogren's syndrome, Sydenham's chorea, Takayasu's arteritis,Wegener's granulomatosis, autoimmune gastritis, autoimmune hepatitis,cutaneous autoimmune diseases, autoimmune dilated cardiomyopathy,multiple sclerosis, myocarditis, myasthenia gravis, pernicious anemia,polymyalgia, psoriasis, rapidly progressive glomerulonephritis,rheumatoid arthritis, ulcerative colitis, vasculitis, autoimmunediseases of the muscle, autoimmune diseases of the testis, autoimmunediseases of the ovary and autoimmune diseases of the eye, acne vulgari,asthma, autoimmune diseases, celiac disease, chronic prostatitis,glomerulonephritis, hypersensitivities, inflammatory bowel diseases,pelvic inflammatory disease, peperfusion injury, rheumatoid arthritis,sarcoidosis, transplant rejection, vasculitis, and interstitialcystitis.

In one aspect, the disclosure provides methods for administering any oneof antibodies or compositions described herein to a subject in needthereof. In some embodiments, a subject in need of treatments is asubject with a disease characterized by a dysregulation of TNF levels.Disease characterized by a dysregulation of TNF levels, in addition tothe inflammatory and immune disorders discussed above, includeAlzheimer, cancer and depression.

In one aspect, the disclosure provides pharmaceutical compositions whichcomprise an amount of an antibody or population of antibodies and apharmaceutically acceptable vehicle, diluent or carrier. In someembodiments, the compositions comprise milk.

In one aspect, the disclosure provides a method of treating a subject,comprising administering to a subject a composition provided in anamount effective to treat a disease the subject has or is at risk ofhaving is provided. In one embodiment the subject is a human. In anotherembodiment the subject is a non-human animal, e.g., a dog, cat, horse,cow, pig, sheep, goat or primate.

According to embodiments that involve administering to a subject in needof treatment a therapeutically effective amount of the antibodies asprovided herein, “therapeutically effective” or “an amount effective totreat” denotes the amount of antibody or of a composition needed toinhibit or reverse a disease condition (e.g., to treat theinflammation). Determining a therapeutically effective amountspecifically depends on such factors as toxicity and efficacy of themedicament. These factors will differ depending on other factors such aspotency, relative bioavailability, patient body weight, severity ofadverse side-effects and preferred mode of administration. Toxicity maybe determined using methods well known in the art. Efficacy may bedetermined utilizing the same guidance. Efficacy, for example, can bemeasured by a decrease in inflammation. A pharmaceutically effectiveamount, therefore, is an amount that is deemed by the clinician to betoxicologically tolerable, yet efficacious.

Dosage may be adjusted appropriately to achieve desired drug (e.g.,anti-TNF-alpha antibodies) levels, local or systemic, depending upon themode of administration. In the event that the response in a subject isinsufficient at such doses, even higher doses (or effective higher dosesby a different, more localized delivery route) may be employed to theextent that patient tolerance permits. Multiple doses per day arecontemplated to achieve appropriate systemic levels of antibodies.Appropriate systemic levels can be determined by, for example,measurement of the patient's peak or sustained plasma level of the drug.“Dose” and “dosage” are used interchangeably herein.

In some embodiments, the amount of antibody or pharmaceuticalcomposition administered to a subject is 50 to 500 mg/kg, 100 to 400mg/kg, or 200 to 300 mg/kg per week. In one embodiment the amount ofantibody or pharmaceutical composition administered to a subject is 250mg/kg per week. In some embodiments, an initial dose of 400 mg/kg isadministered a subject the first week, followed by administration of 250mg/kg to the subject in subsequent weeks. In some embodiments theadministration rate is less than 10 mg/min. In some embodiments,administration of the antibody or pharmaceutical composition to asubject occurs at least one hour prior to treatment with anothertherapeutic agent. In some embodiments, a pre-treatment is administeredprior to administration of the antibody or pharmaceutical composition.

In some embodiments the compositions provided are employed for in vivoapplications. Depending on the intended mode of administration in vivothe compositions used may be in the dosage form of solid, semi-solid orliquid such as, e.g., tablets, pills, powders, capsules, gels,ointments, liquids, suspensions, or the like. Preferably, thecompositions are administered in unit dosage forms suitable for singleadministration of precise dosage amounts. The compositions may alsoinclude, depending on the formulation desired, pharmaceuticallyacceptable carriers or diluents, which are defined as aqueous-basedvehicles commonly used to formulate pharmaceutical compositions foranimal or human administration. The diluent is selected so as not toaffect the biological activity of the human recombinant protein ofinterest. Examples of such diluents are distilled water, physiologicalsaline, Ringer's solution, dextrose solution, and Hank's solution. Thesame diluents may be used to reconstitute a lyophilized recombinantprotein of interest. In addition, the pharmaceutical composition mayalso include other medicinal agents, pharmaceutical agents, carriers,adjuvants, nontoxic, non-therapeutic, non-immunogenic stabilizers, etc.Effective amounts of such diluent or carrier are amounts which areeffective to obtain a pharmaceutically acceptable formulation in termsof solubility of components, biological activity, etc. In someembodiments the compositions provided herein are sterile.

Administration during in vivo treatment may be by any number of routes,including oral, parenteral, intramuscular, intranasal, sublingual,intratracheal, inhalation, ocular, vaginal, and rectal. Intracapsular,intravenous, and intraperitoneal routes of administration may also beemployed. The skilled artisan recognizes that the route ofadministration varies depending on the disorder to be treated. Forexample, the compositions or antibodies herein may be administered to asubject via oral, parenteral or topical administration. In oneembodiment, the compositions or antibodies herein are administered byintravenous infusion.

The compositions, when it is desirable to deliver them systemically, maybe formulated for parenteral administration by injection, e.g., by bolusinjection or continuous infusion. Formulations for injection may bepresented in unit dosage form, e.g., in ampoules or in multi-dosecontainers, with an added preservative. The compositions may take suchforms as suspensions, solutions or emulsions in oily or aqueousvehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents.

Pharmaceutical formulations for parenteral administration includeaqueous solutions of the active compositions in water soluble form.Additionally, suspensions of the active compositions may be prepared asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils such as sesame oil, or synthetic fatty acidesters, such as ethyl oleate or triglycerides, or liposomes. Aqueousinjection suspensions may contain substances which increase theviscosity of the suspension, such as sodium carboxymethyl cellulose,sorbitol, or dextran. Optionally, the suspension may also containsuitable stabilizers or agents which increase the solubility of thecompositions to allow for the preparation of highly concentratedsolutions. Alternatively, the active compositions may be in powder formfor constitution with a suitable vehicle, e.g., sterile pyrogen-freewater, before use.

For oral administration, the pharmaceutical compositions may take theform of, for example, tablets or capsules prepared by conventional meanswith pharmaceutically acceptable excipients such as binding agents(e.g., pregelatinised maize starch, polyvinylpyrrolidone orhydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystallinecellulose or calcium hydrogen phosphate); lubricants (e.g., magnesiumstearate, talc or silica); disintegrants (e.g., potato starch or sodiumstarch glycolate); or wetting agents (e.g., sodium lauryl sulphate). Thetablets may be coated by methods well known in the art. Liquidpreparations for oral administration may take the form of, for example,solutions, syrups or suspensions, or they may be presented as a dryproduct for constitution with water or other suitable vehicle beforeuse. Such liquid preparations may be prepared by conventional means withpharmaceutically acceptable additives such as suspending agents (e.g.,sorbitol syrup, cellulose derivatives or hydrogenated edible fats);emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles(e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetableoils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates orsorbic acid). The preparations may also contain buffer salts, flavoring,coloring and sweetening agents as appropriate. The component orcomponents may be chemically modified so that oral delivery of theantibodies is efficacious. Generally, the chemical modificationcontemplated is the attachment of at least one molecule to theantibodies, where said molecule permits (a) inhibition of proteolysis;and (b) uptake into the blood stream from the stomach or intestine. Alsodesired is the increase in overall stability of the antibodies andincrease in circulation time in the body. Examples of such moleculesinclude: polyethylene glycol, copolymers of ethylene glycol andpropylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol,polyvinyl pyrrolidone and polyproline. Abuchowski and Davis, 1981,“Soluble Polymer-Enzyme Adducts” In: Enzymes as Drugs, Hocenberg andRoberts, eds., Wiley-Interscience, New York, N.Y., pp. 367-383; Newmark,et al., 1982, J. Appl. Biochem. 4:185-189. Other polymers that could beused are poly-1,3-dioxolane and poly-1,3,6-tioxocane. Preferred forpharmaceutical usage, as indicated above, are polyethylene glycolmolecules. For oral compositions, the location of release may be thestomach, the small intestine (the duodenum, the jejunum, or the ileum),or the large intestine. One skilled in the art has availableformulations which will not dissolve in the stomach, yet will releasethe material in the duodenum or elsewhere in the intestine. Preferably,the release will avoid the deleterious effects of the stomachenvironment, either by protection of the antibody or by release of thebiologically active material beyond the stomach environment, such as inthe intestine.

For buccal administration, the compositions may take the form of tabletsor lozenges formulated in conventional manner.

For administration by inhalation, the compositions for use according tothe present disclosure may be conveniently delivered in the form of anaerosol spray presentation from pressurized packs or a nebulizer, withthe use of a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol the dosage unitmay be determined by providing a valve to deliver a metered amount.Capsules and cartridges of e.g. gelatin for use in an inhaler orinsufflator may be formulated containing a powder mix of thecompositions and a suitable powder base such as lactose or starch.

Also contemplated herein is pulmonary delivery. The compositions can bedelivered to the lungs of a mammal while inhaling and traverses acrossthe lung epithelial lining to the blood stream. Contemplated for use inthe practice of this disclosure are a wide range of mechanical devicesdesigned for pulmonary delivery of therapeutic products, including butnot limited to nebulizers, metered dose inhalers, and powder inhalers,all of which are familiar to those skilled in the art.

Nasal delivery of a pharmaceutical composition disclosed herein is alsocontemplated. Nasal delivery allows the passage of a pharmaceuticalcomposition of the present disclosure to the blood stream directly afteradministering the therapeutic product to the nose, without the necessityfor deposition of the product in the lung. Formulations for nasaldelivery include those with dextran or cyclodextran.

The compositions may also be formulated in rectal or vaginalcompositions such as suppositories or retention enemas, e.g., containingconventional suppository bases such as cocoa butter or other glycerides.

The pharmaceutical compositions also may comprise suitable solid or gelphase carriers or excipients. Examples of such carriers or excipientsinclude but are not limited to calcium carbonate, calcium phosphate,various sugars, starches, cellulose derivatives, gelatin, and polymerssuch as polyethylene glycols.

Suitable liquid or solid pharmaceutical preparation forms are, forexample, aqueous or saline solutions for inhalation, microencapsulated,encochleated, coated onto microscopic gold particles, contained inliposomes, nebulized, aerosols, pellets for implantation into the skin,or dried onto a sharp object to be scratched into the skin. Thepharmaceutical compositions also include granules, powders, tablets,coated tablets, (micro)capsules, suppositories, syrups, emulsions,suspensions, creams, drops or preparations with protracted release ofactive compositions, in whose preparation excipients and additivesand/or auxiliaries such as disintegrants, binders, coating agents,swelling agents, lubricants, flavorings, sweeteners or solubilizers arecustomarily used as described above. The pharmaceutical compositions aresuitable for use in a variety of drug delivery systems. For a briefreview of methods for drug delivery, see Langer, Science 249:1527-1533,1990, which is incorporated herein by reference.

The antibodies and optionally other therapeutics may be administered perse (neat) or in the form of a pharmaceutically acceptable salt. Whenused in medicine the salts should be pharmaceutically acceptable, butnon-pharmaceutically acceptable salts may conveniently be used toprepare pharmaceutically acceptable salts thereof. Such salts include,but are not limited to, those prepared from the following acids:hydrochloric, hydrobromic, sulphuric, nitric, phosphoric, maleic,acetic, salicylic, p-toluene sulphonic, tartaric, citric, methanesulphonic, formic, malonic, succinic, naphthalene-2-sulphonic, andbenzene sulphonic. Also, such salts can be prepared as alkaline metal oralkaline earth salts, such as sodium, potassium or calcium salts of thecarboxylic acid group.

Suitable buffering agents include: acetic acid and a salt (1-2% w/v);citric acid and a salt (1-3% w/v); boric acid and a salt (0.5-2.5% w/v);and phosphoric acid and a salt (0.8-2% w/v). Suitable preservativesinclude benzalkonium chloride (0.003-0.03% w/v); chlorobutanol (0.3-0.9%w/v); parabens (0.01-0.25% w/v) and thimerosal (0.004-0.02% w/v).

The pharmaceutical compositions of the disclosure contain an effectiveamount of the antibodies and optionally therapeutic agents included in apharmaceutically-acceptable carrier. The termpharmaceutically-acceptable carrier means one or more compatible solidor liquid filler, diluents or encapsulating substances which aresuitable for administration to a human or other vertebrate animal. Theterm carrier denotes an organic or inorganic ingredient, natural orsynthetic, with which the active ingredient is combined to facilitatethe application. The components of the pharmaceutical compositions alsoare capable of being commingled with the compositions of the presentdisclosure, and with each other, in a manner such that there is nointeraction which would substantially impair the desired pharmaceuticalefficiency.

The therapeutic agent(s), including specifically but not limited to theantibodies, may be provided in particles. Particles as used herein meansnano or microparticles (or in some instances larger) which can consistin whole or in part of the antibody or other therapeutic agentsadministered with the antibody. The particle may include, in addition tothe therapeutic agent(s), any of those materials routinely used in theart of pharmacy and medicine, including, but not limited to, erodible,nonerodible, biodegradable, or nonbiodegradable material or combinationsthereof. The particles may be microcapsules which contain the antibodyin a solution or in a semi-solid state. The particles may be ofvirtually any shape.

Methods of Production of Antibodies

In one aspect, the disclosure provides methods for production of highlygalactosylated anti-TNF-alpha antibodies and populations with highlevels of galactosylated antibodies.

In one aspect, the disclosure provides a method for producing apopulation of antibodies, comprising: expressing the population ofantibodies in mammary gland epithelial cells of a non-human mammal suchthat a population of antibodies is produced, wherein the antibody is ananti-TNF-alpha antibody, and wherein the level of galactosylation of theantibodies in the population is at least 70%. In some embodiments, theanti-TNF-alpha antibody is adalimumab. In some embodiments, the mammarygland epithelial cells are in culture and are transfected with a nucleicacid that comprises a sequence that encodes the antibody. In someembodiments, the nucleic acid comprise SEQ ID NO:3 and SEQ ID NO:4. Insome embodiments, the mammary gland epithelial cells are in a non-humanmammal engineered to express a nucleic acid that comprises a sequencethat encodes the antibody in its mammary gland. In some embodiments, themammary gland epithelial cells are goat, sheep, bison, camel, cow, pig,rabbit, buffalo, horse, rat, mouse or llama mammary gland epithelialcells. In some embodiments, the mammary gland epithelial cells are goatmammary gland epithelial cells.

In one aspect the disclosure provides mammary gland epithelial cellsthat express the highly galactosylated anti-TNF-alpha antibodies orpopulations with high levels of galactosylated antibodies disclosedherein.

In one aspect the disclosure provides a transgenic non-human mammalcomprising mammary gland epithelial cells that express the highlygalactosylated anti-TNF-alpha antibodies or populations with high levelsof galactosylated antibodies disclosed herein.

In one aspect the disclosure provides a method for the production of aglycosylated antibody or population of glycosylated antibodies, theprocess comprising expressing in the milk of a transgenic non-humanmammal a glycosylated antibody encoded by a nucleic acid construct. Inone embodiment the mammalian mammary epithelial cells are of a non-humanmammal engineered to express the antibody in its milk. In yet anotherembodiment the mammalian mammary epithelial cells are mammalian mammaryepithelial cells in culture.

In another embodiment the method comprises:

-   -   (a) providing a non-human transgenic mammal engineered to        express an antibody,    -   (b) expressing the antibody in the milk of the non-human        transgenic mammal;    -   (c) isolating the antibodies expressed in the milk; and    -   (d) detecting the presence galactose on the isolated antibodies.

In yet another embodiment the method, comprises: producing a populationof glycosylated antibodies in mammary gland epithelial cells such thatthe population of glycosylated antibodies produced comprises a specificpercentage of galactosylation (e.g., at least 70%, at least 80%, atleast 90%, or higher). In some embodiment, the antibody is ananti-TNF-alpha antibody. In some embodiments, the glycosylatedantibodies comprise a heavy chain comprising SEQ ID NO:1 and a lightchain comprising SEQ ID NO:2. In some embodiments, this method isperformed in vitro. In other embodiments, this method is performed invivo, e.g., in the mammary gland of a transgenic goat.

In some embodiments the methods above further comprise steps forinducing lactation. In still other embodiments the methods furthercomprise additional isolation and/or purification steps. In yet otherembodiments the methods further comprise steps for comparing theglycosylation pattern of the antibodies obtained with antibodiesproduced in cell culture, e.g. non-mammary cell culture. In furtherembodiments, the methods further comprise steps for comparing theglycosylation pattern of the antibodies obtained to antibodies producedby non-mammary epithelial cells. Such cells can be cells of a cellculture. In some embodiments, the glycosylation pattern is the amount ofgalactose present on an antibody or population of antibodies. In someembodiments, the method further comprises comparing the percentage ofgalactosylation present in the population of glycosylated antibodies tothe percentage of galactosylation present in a population ofglycosylated antibodies produced in cell culture, e.g. non-mammary cellculture. Experimental techniques for assessing the glycosylation patternof the antibodies can be any of those known to those of ordinary skillin the art or as provided herein, such as below in the Examples. Suchmethods include, e.g., liquid chromatography mass spectrometry, tandemmass spectrometry, and Western blot analysis.

The antibodies can be obtained, in some embodiments, by collecting theantibodies from the milk of a transgenic animal produced as providedherein or from an offspring of said transgenic animal. In someembodiments the antibodies produced by the transgenic mammal is producedat a level of at least 1 gram per liter of milk produced.Advantageously, the method according to the invention allows theproduction of more than 4 grams per liter of milk produced,advantageously more than 5, 10, 15, 20, 25, 30, 35, grams per liter,advantageously up to 70 grams per liter.

Unless otherwise defined herein, scientific and technical terms used inconnection with the present disclosure shall have the meanings that arecommonly understood by those of ordinary skill in the art. Further,unless otherwise required by context, singular terms shall includepluralities and plural terms shall include the singular. The methods andtechniques of the present disclosure are generally performed accordingto conventional methods well-known in the art. Generally, nomenclaturesused in connection with, and techniques of biochemistry, enzymology,molecular and cellular biology, microbiology, genetics and protein andnucleic acid chemistry and hybridization described herein are thosewell-known and commonly used in the art. The methods and techniques ofthe present disclosure are generally performed according to conventionalmethods well known in the art and as described in various general andmore specific references that are cited and discussed throughout thepresent specification unless otherwise indicated.

The present invention is further illustrated by the following Examples,which in no way should be construed as further limiting. The entirecontents of all of the references (including literature references,issued patents, published patent applications, and co pending patentapplications) cited throughout this application are hereby expresslyincorporated by reference.

EXAMPLES Materials and Methods

Generation of Transgenic Goats that Produce Adalimumab Transgenic goatswere generated that include the nucleic acid sequence encoding theadalimumab antibody in their genome. The goats producing adalimumab weregenerated using traditional micoinjection techniques (See e.g., U.S.Pat. No. 7,928,064). The cDNA encoding the heavy and light chain (SEQ IDNO:3 and SEQ ID NO:4) was synthesized based on the published amino acidsequence (U.S. Pat. No. 6,090,382). These DNA sequences were ligatedwith the beta casein expression vector to yield constructs BC2601 HC andBC2602 LC. In these plasmids, the nucleic acid sequence encodingadalimumab is under the control of a promoter facilitating theexpression of adalimumab in the mammary gland of the goats. Theprokaryotic sequences were removed and the DNA microinjected intopre-implantation embryos of the goat. These embryos were thentransferred to pseudo pregnant females. The progeny that resulted werescreened for the presence of the transgenes. Those that carried bothchains were identified as transgenic founders.

When age appropriate, the founder animals were bred. Following pregnancyand parturition they were milked. The time course was in days startinglactation after parturation (e.g., day 3, day 7, day 11). The adalimumabantibody was purified from the milk at each time point and characterizedas described herein.

Measuring the Binding of Transgenically Produced Adalimumab by ELISA:

5 μg/ml of TNF-α was coated overnight at 4° C. in a 96 well plate in 100μl of PBS per well. After blocking of nonspecific sites (incubation with200 μl PBS/1% BSA, 1 h RT), transgenically produced adalimumab ordeglycosylated adalimumab was added at various concentrations (0 to 10μg/ml) for 20 min in PBS/1% BSA. After washing, the binding oftransgenically produced adalimumab to coated TNF-α was evaluated by theaddition of a goat anti-human IgG (H+L) coupled to peroxidase, followedby substrate (H₂O₂ and tetramethylbenzidine). After 20 min ofincubation, the reaction was stopped with 50 μl of diluted H₂SO₄ and theOD was read at 450 nm. The results for transgenically producedadalimumab are shown in FIG. 10.

Binding to CD16, Competition of 3G8 Antibody

To evaluate the binding of transgenically produced adalimumab to CD16, adisplacement study with the anti-CD16 binding antibody 3G8, (Santa CruzBiotech) was performed. The displacement test allowed for thedetermination of the binding of the transgenically produced adalimumabto the CD16 receptor expressed at NK surface membrane.

Natural killer cells (NK cells) were purified by negative depletion(Miltenyi) from the peripheral blood of healthy donors. The NK cellswere then incubated with variable concentrations (0 to 83 μg/ml) oftransgenically produced adalimumab and the anti-CD16 antibody 3G8conjugated to a fluorochrome (3G8-PE), at a fixed concentration. Afterwashing, the binding of 3G8-PE to the CD16 receptor on the NK cells wasevaluated by flow cytometry. The mean fluorescence values (MFI) observedwere expressed as the percent binding, where a value of 100% correspondsthe value observed without the tested transgenically produced adalimumabthat thus corresponds to maximum 3G8 binding. A value of 0% correspondsto the MFI in the absence of the antibody 3G8. IC₅₀, the antibodyconcentration required to induce inhibition of 3G8 binding by 50% ofImax, were calculated using PRISM software. The results are shown inFIG. 11.

Binding of Soluble TNF-α with Transgenically Produced Adalimumab on CD16Expressed by Jurkat Cells Via the Fc Fragment of Transgenically ProducedAdalimumab

Jurkat-CD16 cells were incubated with 10 μg/ml of transgenicallyproduced adalimumab or the deglycosylated version thereof for 20 min at4° C. After washing, 100 μl of TNF-α was added to the cell pellet at afinal concentration of 1 μg/ml, 20 min at 4° C. After subsequentwashing, the cells were incubated with 5 μg/ml of a biotinylated goatanti-human TNF-α antibody, 20 min at 4° C. After another round ofwashing, the binding of TNF-α was visualized by the addition ofstreptavidin coupled to PE-fluorochrome for 20 min at 4° C. Samples wereanalyzed by flux cytometry. The results are shown in FIG. 12.

Example 1: Transgenically Produced Adalimumab

The glycosylation pattern of the adalimumab antibodies produced in themilk of transgenic goats was determined by releasing the N-glycans fromantibody and running the released oligosaccharides on a column(“oligosaccharide signature”).

FIGS. 1-4 and 6-8 show the N-glycan oligosaccharides released from thetransgenically produced adalimumab antibody from goat #1 (FIGS. 1-4) andgoat #2 (FIGS. 6-8). The monosaccharide groups are depicted as follows:

-   -   Black square: N-AcetylGlucosamine (GlcNac)    -   Triangle: Fucose    -   Grey Circle: Mannose    -   White Circle: Galactose    -   Grey Diamond: N-GlycolylNeuraminic Acid (NGNA): a sialic acid    -   White Diamond: N-AcetylNeuraminic Acid (NANA): a sialic acid

FIG. 1 shows a representative chromatogram of N-glycan oligosaccharidesreleased from the transgenic adalimumab antibody produced in the milk ofgoat #1. The chromatogram shows that of the fourteen major N-glycanoligosaccharides produced, twelve have at least one galactose in theN-glycan chain, with four oligosaccharides having two galactoses. Onlytwo of the oligosaccharides are purely oligomannose (See peak 1 and peak3). FIG. 1 also shows that of the fourteen major oligosaccharidesproduced, nine are fucosylated All of the fucosylated oligosaccharidesare also galactosylated.

FIGS. 2-4 show chromatograms of N-glycan oligosaccharides released fromthe transgenic adalimumab antibody produced in the milk of goat #1 asharvested after 7 days of lactation (FIG. 2), 17 days of lactation (FIG.3), and 32 days of lactation (FIG. 4).

The relative percentages of all N-glycan oligosaccharides isolated fromthe adalimumab antibody produced in the milk of goat #1 are depicted inFIG. 5. FIG. 5 also shows a tabulation of the overall percentage ofmono-galactosylation, percentage of bi-galactosylation, percentage oftotal galactosylation (mono-galactosylation+bi-galactosylation),percentage of galactosylation as calculated according to the formulaprovided above, percentage of fucosylation as calculated according tothe formula provided above, the ratio of galactosylation to fucosylationand the percentage of glycan structures with at least one sialic acid (%sialylation). The results are also summarized in Table 1 below:

TABLE 1 N-glycan oligosaccharides isolated from adalimumab antibodiesfrom goat #1 day 7 day 17 day 32 average mono-Gal (%): 30.8 42.9 44.139.2 bi-Gal (%): 53.1 46.0 47.0 48.7 mono-Gal + bi-Gal (%) 83.9 88.991.1 88.0 Gal* (%) 82.9 88.2 89.8 87.0 Fuc* (%) 63.5 74.9 81.9 73.4Ratio Gal/Fuc 1.30 1.17 1.10 1.18 Silaylation (%) 50.4 59.3 62.7 57.5*calculated according to formulas in specification

FIGS. 6-8 show chromatograms of N-glycan oligosaccharides released fromthe transgenically produced adalimumab antibody in the milk of goat #2as harvested after 3 days of lactation (FIG. 6), 11 days of lactation(FIG. 7), and 21 days of hormone induced lactation (FIG. 8).

The relative percentages of all N-glycan oligosaccharides isolated fromthe adalimumab antibody produced in the milk of goat #2 are depicted inFIG. 9. FIG. 9 also shows a tabulation of the overall percentage ofmono-galactosylation, percentage of bi-galactosylation, percentage oftotal galactosylation (mono-galactosylation+bi-galactosylation),percentage of galactosylation as calculated according to the formulaprovided above, percentage of fucosylation as calculated according tothe formula provided above, the ratio of galactosylation to fucosylationand the percentage of glycan structures with at least one sialic acid (%sialylation). The results are also summarized below:

TABLE 2 N-glycan oligosaccharides isolated from adalimumab antibodiesfrom goat #2 day 3 day 11 day 21 average mono-Gal (%): 27.3 25.7 27.526.8 bi-Gal (%): 39.0 43.0 31.4 37.8 mono-Gal + bi-Gal (%) 66.3 68.758.9 64.6 Gal* (%) 64.6 67.9 57.8 63.4 Fuc* (%) 51.6 54.0 46.0 50.5Gal/Fuc 1.25 1.25 1.25 1.25 Sialylation (%) 40.8 42.6 39.1 40.8*calculated according to formulas in specification

Example 2: Binding Studies of Transgenically Produced Adalimumab

FIG. 10 shows that transgenically produced adalimumab can bind solubleTNF-alpha coated in 96-well plates. Transgencially produced adalimumabthat was aglycosylated was able to bind soluble TNF-alpha as well (datanot shown).

FIG. 11 shows that transgenically produced adalimumab binds CD16expressed by natural killer (NK) cells. The binding was shown in acompetition experiment with the anti-CD16 binding antibody 3G8. Thebinding of a transgenically produced adalimumab to CD16 is stronger thanthe binding of a poorly galactosylated antibody to CD16 (data notshown). The transgenically produced adalimumab is therefore expected tohave a higher ADCC activity.

FIG. 12 shows that a transgenically produced adalimumab antibody bindsboth soluble TNF-alpha and Jurkat expressing CD16 cells while anaglycosylated version of the transgenically produced adalimumab antibodydoes not. The transgenically produced adalimumab antibody therefore isexpected to show ADCC activity while the aglycosylated antibody doesnot.

Example 3: Glycosylation Analysis of Transgenically Produced Adalimumabin Additional Animals

TABLE 3 Summary of adalimumab production data Est. #Days Average Milkmg/mL % Total Goat Lactation Volume (mL) in WM Agg #mg Purified 1 32  245 29  20 400 (more TBD) 8 36   350 14* 20 1496 9 36   382  35** 253929 3 36*** 634 5 14 502 4 29*** 1458 4 12 390 5 29*** 1663 4 8 341 636*** 1899 4 12 374 7 29*** 1676 5 8 395 2 36   260  34** 3 3719*Concentration started at 20 mg/mL and dropped to 10 mg/mL. **Valuescould be higher if protein A column was overloaded. ***Lactation volumewas still high when dried off.

Adalimumab was transgenically produced in multiple different goatsduring natural lactation. The glycan profiles for transgenicallyproduced adalimumab were examined. FIG. 13 provides a summary ofpercentages of different glycan forms present in populations oftransgenically produced adalimumab antibody from the nine differentgoats listed in Table 3.

FIG. 14 shows a summary of the percentages of N-glycan oligosaccharidesof populations of transgenically produced adalimumab antibodies fromgoat #10 and goat #1 during hormone induced lactation.

FIG. 15 shows a summary of N-glycan oligosaccharides of populations oftransgenically produced adalimumab antibodies from eight differentgoats, goats #2-9. The data presented in FIG. 15 is summarized in Tables4-11.

TABLE 4 N-glycan oligosaccharides isolated from adalimumab antibodiesfrom goat # 2 day 29 mono-Gal (%) 6.9 bi-Gal (%) 11.9 mono-Gal + bi-Gal(%) 18.8 Gal* (%) 16.9 Fuc* (%) 13.2 Ratio Gal/Fuc 1.28

TABLE 5 N-glycan oligosaccharides isolated from adalimumab antibodiesfrom goat # 4 day 29 mono-Gal (%) 23.2 bi-Gal (%) 11.3 mono-Gal + bi-Gal(%) 34.5 Gal* (%) 24.1 Fuc* (%) 30.4 Ratio Gal/Fuc 0.79

TABLE 6 N-glycan oligosaccharides isolated from adalimumab antibodiesfrom goat # 7 day 29 mono-Gal (%) 28.1 bi-Gal (%) 14.3 mono-Gal + bi-Gal(%) 42.4 Gal* (%) 30.7 Fuc* (%) 47 Ratio Gal/Fuc 0.65

TABLE 7 N-glycan oligosaccharides isolated from adalimumab antibodiesfrom goat # 6 day 29 mono-Gal (%) 31.9 bi-Gal (%) 14.3 mono-Gal + bi-Gal(%) 47 Gal* (%) 34.2 Fuc* (%) 49.6 Ratio Gal/Fuc 0.69

TABLE 8 N-glycan oligosaccharides isolated from adalimumab antibodiesfrom goat # 3 day 29 mono-Gal (%) 29.4 bi-Gal (%) 23.6 mono-Gal + bi-Gal(%) 53 Gal* (%) 46.1 Fuc* (%) 52.8 Ratio Gal/Fuc 0.87

TABLE 9 N-glycan oligosaccharides isolated from adalimumab antibodiesfrom goat #9 day 29 mono-Gal (%) 37.7 bi-Gal (%) 44 mono-Gal + bi-Gal(%) 81.7 Gal* (%) 81.7 Fuc* (%) 78.2 Ratio Gal/Fuc 1.04

TABLE 10 N-glycan oligosaccharides isolated from adalimumab antibodiesfrom goat #5 day 29 mono-Gal (%) 26.2 bi-Gal (%) 11.8 mono-Gal + bi-Gal(%) 38.0 Gal* (%) 27.6 Fuc* (%) 39.4 Ratio Gal/Fuc 0.70

TABLE 11 N-glycan oligosaccharides isolated from adalimumab antibodiesfrom goat # 8 day 29 mono-Gal (%) 18.6 bi-Gal (%) 65.4 mono-Gal + bi-Gal(%) 84 Gal* (%) 80.4 Fuc* (%) 74.0 Ratio Gal/Fuc 1.09

Example 4: Characterization of Transgenically Produced Adalimumab

Biological features of transgenically produced adalimumab from goat milkwere compared to Adalimumab (Humira). Relative Kd, CD16-binding and FcRnbinding were investigated. Furthermore, Complement DependentCytotoxicity (CDC) and neutralisation of TNF-α mediated cellularcytotoxicity were evaluated on membrane TNF-α transfected Jurkat cells.

Arbitrary Kd (concentration giving 50% of the plateau value) obtained byflux cytometry showed no difference (p=0.54) between Humira (0.09 μg/ml)and transgenically produced adalimumab (0.11 μg/ml). Transgenicallyproduced adalimumab bound to CD16 receptor with an IC50 value of 25.7μg/ml versus 56.67 μg/ml for Humira. Thus, binding of transgenicallyproduced adalimumab to CD16 was 2.2 fold higher than that of Humira.

Transgenically produced adalimumab binding to FcRn was higher than thatof Humira, despite having the same Fc portion. CDC activity showed anadvantage with transgenically produced adalimumab compared to Humiraantibody (196 vs 399 ng/ml). Transgenically produced adalimumab andHumira both neutralized similarly TNF-α induced L929 apoptosis (EC50=110ng/ml and 98 ng/ml respectively).

Materials and Methods

Reagents

-   -   Anti TNF-α reagent:        -   Antibody:            -   Transgenically produced adalimumab            -   Humira (Adalimumab, Abbott)    -   Anti CD160: purification n° 829 10 049, used as negative control    -   baby rabbit serum (Cederlane)    -   Actinomycin D (Sigma)    -   Human TNF-α (Miltenyi Biotec)    -   Cell titer 96® aqueous One solution Cell Proliferation Assay        (Promega)

Cells

-   -   Membrane TNF-α transfected Jurkat cells clone 2B3 were obtained        by cloning (used for Relative Kd assay and CDC)    -   Murine fibroblast L929 cells

Binding to Membrane TNF-α and Relative Kd Determination

2×10⁵ TNF-α transfected Jurkat cells were incubated with 100 μl ofanti-TNF Alexa-488 coupled antibodies at different concentrations (0 to100 μl/ml, final concentration) at 4° C. for 30 minutes. After washing,a goat anti-human Fc gamma coupled to phycoerythrin (100 μl of adilution of 1:100) was added at 4° C. for 30 minutes. The cells werewashed and mean of fluorescence intensity (MFI) studied by flowcytometry. Arbitrary Kd was calculated using PRISM software.

Binding to CD16

Binding to CD16 was studied by a competitive assay using the mousephycoerythrin-labelled anti-CD16 3G8 (3G8-PE) and NK cells. Briefly, NKcells were isolated from peripheral blood mononuclear cells (PBMC) usingthe NK Cell Isolation Kit from Myltenyi then incubated with variableconcentrations (0 to 83 μg/ml) of the tested antibodies simultaneouslywith the mouse anti-CD16 mAb 3G8-PE used at a fixed concentration. Afterwashing, the binding of 3G8-PE to CD16 expressed by NK cell wasevaluated by flow cytometry. The mean fluorescence values (MFI) observedare expressed in percent, 100% being the value obtained with the 3G8-PEalone and 0% the value in the absence of the 3G8-PE. IC50 values(antibody concentration required to induce 50% inhibition of 3G8binding) were calculated using PRISM software.

Binding to FcRn

Binding to FcRn was studied by a competitive assay using the anti-CD20Rituxan labeled by Alexa 488 (RTX-Alexa) and transfected FcRn Jurkatcells. Briefly, transfected FcRn Jurkat cells were incubated withvariable concentrations (0 to 1000 μg/ml) of the tested mAbssimultaneously with a fixed concentration RTX-Alexa (50 μg/ml) at pH=6.After washing at pH=6, binding of RTX-Alexa to FcRn expressed bytransfected FcRn Jurkat cells was evaluated by flow cytometry. The meanfluorescence values (MFI) observed are expressed in percent, 100% beingthe value obtained with the RTX-Alexa alone and 0% the value in theabsence of the RTX-Alexa. IC50 values (antibody concentration requiredto induce 50% of inhibition of RTX-Alexa binding) were calculated usingPRISM software.

CDC

Membrane TNF-α transfected Jurkat cells (mbTNF-α Jurkat) were incubatedwith increasing concentrations of anti-TNF antibodies (0 to 5000 ng/ml)in the presence of baby rabbit serum as a source of complement (dilutionto 1/10). After 2 hours of incubation at 37° C., the quantity of LDHreleased in the supernatant by the lysed target cells was measured(Roche Applied Sciences Cytotoxicity Detection Kit). The percent lysiscorresponding to the complement-dependent cytotoxicity (CDC) mediated bythe studied antibodies was calculated according to the followingformula: % lysis=(ER−SA), where ER=effective response (LDH release),SA=spontaneous activity obtained when target cell were incubated in thepresence of complement but without antibody. Percent lysis is expressedas a function of antibody concentrations. Emax (percentage of maximumlysis) and EC50 (quantity of antibody that induces 50% of maximum lysis)were calculated using PRISM software.

Neutralization of sTNF-α

Neutralization of human TNF was assessed in the murine fibroblast L929bioassay using a range of concentrations of the anti-TNFs and a fixedconcentration of TNF (100 pg/mL). Briefly, TNF-α (20 ng/ml) pretreatedwith serial dilutions of tested antibodies (0-1000 ng/ml) was incubatedfor 18 h with L929 cells in the presence of actinomycin D (2 μg/ml) at37° C. for 16 h. Then 20 μl/well of Cell titer 96® aqueous One solutionCell Proliferation Assay (MTS) was added for a further 1 h to determinethe number of surviving cells by colorimetric assay. The plates wereread at 490 nm and results (OD) were expressed as a function of antibodyconcentrations. The neutralizing titer expressed as the reciprocal ofthe antibody concentration that neutralizes 50% of TNF-α activity wascalculated using PRISM software.

Abbreviations

TNF-α: Tumor Necrosis Factor alpha

CDC: Complement Dependent Cytotoxicity

LDH: Lactate dehydrogenase

MFI: Mean of Fluorescence Intensity SA: Spontaneous Activity

SR: Spontaneous release

Results Antigen-Binding on Cell and Relative Kd Determination

The mean of three assays is presented in FIG. 16 and the correspondingdata are presented in Table 13. Binding of anti-TNF antibodies tomembrane TNF-α transfected Jurkat cells clone 2B3 is expressed as themean of fluorescence intensity (MFI) for each antibody concentrationtested (0-10 μg/ml). Arbitrary Kd as described below does not representthe real affinity value (nM), but gives an order of comparable magnitudefor the affinity of the studied antibodies.

Results in Table 12a show that the Bmax values (plateau) and arbitraryKd (concentration giving 50% of the plateau value) are similar fortransgenically produced adalimumab (Bmax:MFI=12.46; Kd=0.11 μg/ml) andHumira (Bmax:MFI=10.72; Kd=0.09 μg/ml) as shown with the statisticalanalysis (p=0.54) performed with individual EC50 values (Table 12b).

TABLE 12a Bmax, relative Kd values and original data corresponding toFIG. 16 Transgenically produced Humira adalimumab (Abbott) Anti CD 160Bmax (MFI) 12.46 10.72 na Kd (μg/ml) 0.1194 0.09874 na

TABLE 12b Individual EC50 values and P value (student test)Transgenically produced Humira adalimumab (Abbott) EC50-1 1 5.57142857EC50-2 1 0.09640719 EC50-3 1 0.81034483 p student 0.536862306

TABLE 13 Data corresponding to FIG. 16 m Ab (μg/ml) Transgenicallyproduced adalimumab Humira (Abbott) 0 0.502 1.36 0.816 0.502 1.36 0.8160.01 0.81 1.81 1.69 0.87 1.69 1.63 0.1 2.50 5.80 6.99 3.34 5.66 6.57 0.23.33 9.45 9.86 4.30 8.74 8.41 0.3 3.84 12.90 10.30 4.66 11.50 8.76 0.45.23 14.50 10.60 4.65 11.80 9.19 0.5 5.14 15.50 10.40 4.86 13.10 9.15 16.05 17.20 11.20 5.10 14.10 9.32 2 5.80 17.80 11.30 5.40 15.00 9.76 55.56 18.40 11.60 5.78 15.70 9.85 10 6.30 18.00 12.10 6.33 16.60 9.89

Binding to CD16, Competition of 3G8 Antibody

IC50 values, indicated in Table 14, represent the antibody concentrationrequired to induce 50% of inhibition of 3G8 binding on CD16 receptorexpressed by NK cells. Transgenically produced adalimumab (“TG-Humira”),and Humira antibody bind to CD16 receptor with an IC50 value of 25.7μg/ml and 56.7 μg/ml respectively. Thus, binding of transgenicallyproduced adalimumab to CD16 is almost 2 fold higher than that of Humira.

TABLE 14 IC50 (antibody concentration required to induce 50% ofinhibition of 3G8 binding), corresponding to FIG. 17, after modeling ofthe curve by the PRISM software. Transgenically Humira produced (Abbott)adalimumab IC50 (μg/ml) 57 26

TABLE 15 Data corresponding to FIG. 17 Log C μg/ml Humira (Abbott)Transgenically produced adalimumab 0.01 100 100 100 100 100 100 0.1196.95 89.81 96.44 96.06 95.59 101.36 0.41 99.24 89.81 102.59 94.49 88.9798.98 0.72 97.71 85.35 102.27 88.19 75.74 93.54 1.02 90.08 71.34 99.6878.35 57.06 82.65 1.32 83.21 55.92 91.91 62.76 41.03 71.43 1.62 71.339.94 83.5 45.67 28.31 56.12 1.92 56.95 28.22 67.96 34.65 19.34 42.52

Binding to FcRn

Binding of anti-TNF-α antibodies to FcRn expressed by FcRn-transfectedJurkat cells was tested in a competition assay with Alexa 488coupled-Rituximab antibody. Results showed in FIG. 18 (correspondingdata Table 17) indicate that all the tested antibodies bound to the FcRnreceptor. Binding of transgenically produced adalimumab to FcRn appearedhigher than that of Humira.

TABLE 16 IC50 (antibody concentration required to induce 50% ofinhibition of Rituximab binding), corresponding to FIG. 18, aftermodeling of the curve by the PRISM software. Transgenically producedHumira RITUXAN adalimumab (Abbott) IC50 (μg/ml) 233 195 366

TABLE 17 Data corresponding to FIG. 18 Ac ng/ml (Log) Transgenicallyproduced adalimumab Humira (Abbott) −1 96.6 93.75 92.4 107.4 101.25 1140.602 112.7 84.75 96 118.3 86.25 132 0.903 117.8 81 94.7 135.5 85.5138.2 1.204 113.2 73.65 90.2 125.4 85.5 144 1.505 111.1 66.675 89.5123.6 78.75 144.3 1.806 99.9 64.125 83.7 114.1 65.625 125.3 2.097 94.351.825 72.8 95 58.425 104 2.398 67.4 33.9 53.1 75 45.525 89.2 2.699 39.822.875 35.4 50.6 32.925 64.1

CDC Activity

The mean of two assays is presented in FIG. 19 and the correspondingdata are shown in Table 18. The CDC activity is expressed as the percentof lysis for each antibody concentration tested (0-5000 ng/ml). FIG. 19shows that transgenically produced adalimumab (TG-Humira; maximal lysis:34%) and Humira (maximal lysis: 33%) antibodies induce a comparablelysis of mbTNF-α Jurkat by CDC activity). Analysis of EC50 values inthis system (Table 19) showed a small advantage of transgenicallyproduced adalimumab compared to benchmark Humira antibody (195.8 vs399.3 ng/ml)

TABLE 18 Original data from each of the three experiments performed withthe antibodies tested and used for designing FIG. 19 with PRISMsoftware. Ac ng/ml (Log) Transgenically produced adalimumab Humira(Abbott) −1 0 0 0 0 0 0 0 0 1.6 0 6 0 10 0 2 0 10 1.9 0 10 0 21 0 2 0 192.2 6 20 9 30 1 10 3 23 2.5 11 30 13 35 6 23 8 24 2.8 18 43 16 35 13 2511 31 3.1 19 37 20 38 14 30 16 39 3.4 23 42 17 44 13 38 21 45 3.7 25 4823 48 16 36 22 48

TABLE 19 Emax (maximal lysis) and EC50 (antibody concentration requiredto induce 50% of maximal lysis), corresponding to FIG. 19, after sigmoidmodeling of the curve by the PRISM software. Transgenically producedHumira adalimumab (Abbott) anti-CD160 Emax (% of lysis) 34.31 33.142.839 EC50 (ng/ml) 195.8 399.3 na

Neutralization Activity of Anti-TNF Antibodies

As shown in FIG. 20, TNF-α-mediated cytotoxicity in L929 cells treatedwith 20 ng/ml of human TNF-α was effectively neutralized by bothtransgenically produced adalimumab (TG-humira) and Humira mAbs in acomparable dose dependent manner. The 50% values, corresponding to theantibody concentration required to achieve 50% of the Humira plateauvalue, were 97.7 ng/ml for Humira and 109.7 ng/ml for transgenicallyproduced adalimumab.

TABLE 20 Maximal neutralization and 50% value (antibody concentrationrequired to achieve 50% of the Humira plateau value), corresponding toFIG. 20, after sigmoid modeling of the curve by the PRISM software.Transgenically produced Humira adalimumab (Abbott) % neutralization max66.2 54.6 50% of Humira 109.65 97.72 neutralization (ng/ml)

TABLE 21 Original data from each of the three experiments performed withthe antibodies tested and used for designing FIG. 20 with PRISMsoftware. % TNF neutralization Ac ng/ml (Log) Transgenically producedadalimumab Humira (Abbott) 1 0 2.17 2.58 0 0 1.24 0.47 0 1.4 0 0.71 1.840 0.25 0.73 0.7 0 1.7 0 1.56 2.01 0 0.17 0.45 1.22 5.71 1.9 0.17 7.085.87 19.93 0.88 5.7 11.69 19.25 2 4.35 32.77 13.15 47.85 6.98 21.3942.07 55.18 2.2 31.78 63.85 54.7 56.47 42.01 58.88 40.68 59.71 2.3 49.6476.17 63.88 55.87 52.04 59.01 43.6 60.98 2.8 54.62 88.3 46.78 75.9460.47 68.4 39.53 60.68

Discussion

Neutralisation of TNF induced L929 apoptosis was comparable betweentransgenically produced adalimumab and Humira (Abott). CDC activity,binding to CD16 and FcRn was improved and increased CDC and CD16 bindingwere observed for transgenically produced adalimumab.

Example 5: ADCC Activity of Transgenically Produced Adalimumab

ADCC activity of transgenically produced adalimumab was compared to ADCCactivity of Humira. FIG. 21 shows that transgenically producedadalimumab demonstrated higher ADCC activity than Humira at threedifferent concentrations: 0.5 ng/ml, 50 ng/ml and 5000 ng/ml.

-   -   Antibodies: -Humira (Abbott)        -   Transgenically produced adalimumab    -   Cells: -mTNF-transfected Jurkat cells (clone 2F8)        -   NK effector cells isolated from a healthy donors

NK effector cells were isolated from the peripheral blood samples fromhealthy donors using a negative depletion kit (Miltenyi Biotec). Jurkatcells transfected with mTNF were plated in 96 well plates and incubatedat 4° C. with increasing concentrations of anti-TNF antibodies (0,5; 50and 5000 ng/ml). After 2 hours, the isolated NK cells were added andincubated for another 4 hours at 37° C. Lysis of target cells induced bythe tested anti-TNF antibodies was measured chromogenically byquantifying the intracellular enzyme lactate dehydrogenase (LDH)released into the supernatant by the lysed target cells (Roche). Thepercent lysis was calculated according to the following formula:

% lysis=[(ER−SR)/(100−SR)]−[(NC−SR)/(100−SR)]

Where ER and SR represent experimental and spontaneous LDH release,respectively, and NC represents natural cytotoxicity. Final results areexpressed in arbitrary units with 100% as the value obtained with Humira(Abbott) at a concentration of 5000 ng/ml. As presented in FIG. 21transgenically produced adalimumab induced more ADCC than Humira(Abbott).

Example 6: Assessment of CD16-Binding and CDC Activity of TransgenicallyProduced Adalimumab

CD16 binding activity of transgenically produced adalimumab was measuredby a competitive assay using NK cells and compared to Humira. FIG. 22presents an inhibition curve showing that transgenically producedadalimumab from nine different goats all bound to CD16 to a greaterextent than Humira in NK cells. FIG. 23 presents IC50 values from CD16competitive binding assays. IC50 values were 1.5-5× lower fortransgenically produced adalimumab than for Humira.

CDC activity of transgenically produced adalimumab was compared to thatof Humira. FIG. 24 presents a dose response curve showing thattransgenically produced adalimumab from eight out of nine goats testedhad greater CDC activity than Humira. FIG. 25 presents the EC50 valuesin ng/ml for the CDC activity assay.

Other Embodiments

All of the features disclosed in this specification may be combined inany combination. Each feature disclosed in this specification may bereplaced by an alternative feature serving the same, equivalent, orsimilar purpose. Thus, unless expressly stated otherwise, each featuredisclosed is only an example of a generic series of equivalent orsimilar features.

From the above description, one skilled in the art can easily ascertainthe essential characteristics of the present invention, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various usages andconditions. Thus, other embodiments are also within the claims.

What is claimed is: 1-77. (canceled)
 78. A transgenic non-human mammalcomprising mammary gland epithelial cells, wherein the mammary glandepithelial cells of the transgenic non-human mammal comprise cells thatare genetically engineered to express a nucleic acid sequence encodingthe heavy chain of the anti-TNF-alpha antibody and a nucleic acidsequence encoding the light chain of an anti-TNF-alpha antibody.
 79. Thetransgenic non-human mammal of claim 78, wherein the nucleic acidsequence encoding the heavy chain of the anti-TNF-alpha antibodycomprises a nucleic acid sequence of SEQ ID NO: 3, and wherein thenucleic acid sequence encoding the light chain of the anti-TNF-alphaantibody comprises a nucleic acid sequence of SEQ ID NO:
 4. 80. Thetransgenic non-human mammal of claim 79, wherein the nucleic acidsequence encoding the heavy chain of the anti-TNF-alpha antibody and thenucleic acid sequence encoding the light chain of the anti-TNF-alphaantibody are physically linked.
 81. The transgenic non-human mammal ofclaim 79, wherein the nucleic acid sequence encoding the heavy chain ofthe anti-TNF-alpha antibody and the nucleic acid sequence encoding thelight chain of the anti-TNF-alpha antibody are operably linked to a milkspecific promoter.
 82. The transgenic non-human mammal of claim 81,wherein the milk specific promoter is a casein promoter, a b-caseinpromoter, a r-casien promoter, a K-casein promoter, a whey acidicprotein promoter, or a α-lactalbumin promoter.
 83. The transgenicnon-human mammal of claim 79, wherein the non-human mammal is a goat, asheep, a bison, a camel, a cow, a pig, a rabbit, a buffalo, a horse, arat, a mouse or a llama.
 84. The transgenic non-human mammal of claim83, wherein the non-human mammal is a goat.
 85. The transgenic non-humanmammal of claim 83, wherein the mammary gland epithelial cells expressesa population of anti-TNF-alpha antibodies.
 86. The transgenic non-humanmammal of claim 85, wherein the level of galactosylation of thepopulation of anti-TNF-alpha antibodies is at least 70%.
 87. Thetransgenic non-human mammal of claim 85, wherein the population ofanti-TNF-alpha antibodies comprises mono-galactosylated N-glycans and/orbi-galactosylated N-glycans.
 88. The transgenic non-human mammal ofclaim 86, wherein the level of fucosylation of the population ofanti-TNF-alpha antibodies is at least 50%.
 89. The transgenic non-humanmammal of claim 88, wherein the ratio of the level of galactosylation tothe level of fucosylation of the population of anti-TNF-alpha antibodiesis between 1.0 and 1.4.
 90. The transgenic non-human mammal of claim 89,wherein at least 35% of the antibodies in the population ofanti-TNF-alpha antibody comprise bi-galactosylated N-glycans and atleast 25% of the antibodies in the population of anti-TNF-alphaantibodies comprises mono-galactosylated N-glycans.
 91. The transgenicnon-human mammal of claim 85, wherein the population of anti-TNF-alphaantibodies has an increased level of complement dependent cytotoxicity(CDC) activity when compared to a population of antibodies not producedin mammary gland epithelial cells.
 92. The transgenic non-human mammalof claim 85, wherein the population of anti-TNF-alpha antibodies has anincreased level of antibody-dependent cellular cytotoxicity (ADCC)activity when compared to a population of antibodies not produced inmammary gland epithelial cells.
 93. The transgenic non-human mammal ofclaim 79, wherein the anti-TNF-alpha antibody comprises a heave chain,which comprises an amino acid sequence of SEQ ID NO: 1, and a lightchain, which comprises an amino acid sequence of SEQ ID NO:
 2. 94. Thetransgenic non-human mammal of claim 85, wherein at least 30% of theantibodies in the population of antibodies contain at least oneoligomannose; and wherein the level of fucosylation of the antibodies inthe population of antibodies is at least 30%.
 95. The transgenicnon-human mammal of 94, wherein the antibodies in the population ofantibodies exhibit a high mannose glycosylation pattern.
 96. Thetransgenic non-human mammal of claim 95, wherein at least one chain ofthe antibodies in the population of antibodies contains an oligomannoseand is non-fucosylated.
 97. The transgenic non-human mammal of claim 94,wherein the major carbohydrate of the antibodies in the population ofantibodies is non-fucosylated.